BIOLOGY  LIBRARY 


PROBLEMS  IN  ANIMAL 
METABOLISM 

A    COURSE     OF     LECTURES     GIVEN     IN    THE 

PHYSIOLOGICAL       LABORATORY       OF       THE 

LONDON  UNIVERSITY  AT  SOUTH  KENSINGTON 

IN  THE  SUMMER  TERM,  1904 


BY    J.    B.    LEATHES 

M 

LECTURER    ON    PHYSIOLOGY    IN    THE    MEDICAL   SCHOOL   OF   ST   THOMAS'S    HOSPITAL 


OFTHE          * 

UNIVERSITY 

OF 


PHILADELPHIA 

P.  BLAKISTON'S  SON  AND  CO. 

1012  WALNUT  STREET 

1906 


flu< 

Printed  in  Great  Britain 


, 


if' 


UBKARY 


PREFACE 


THE  subject  chosen  for  these  lectures  was,  I  think,  sufficiently 
indicated  in  the  title  under  which  they  were  announced.  The 
underlying  motive  in  current  investigations  is  frequently  the 
solution  of  problems  which  are  not  expressly  formulated.  Such 
problems  are  certainly  not  ripe  for  dogmatic  treatment.  But 
the  lectures  in  the  University  Laboratory  at  South  Kensington 
are  intended  for  those  students  of  medicine  who  have  already 
had  the  opportunity  of  learning  the  essential  facts  in  physiology. 
They  are  planned  to  foster  in  those  studying  the  practice  of 
medicine  an  interest  in  the  efforts  of  others  who  study  the 
theory,  and  to  keep  alive  the  faith  that  among  the  theories  of 
to-day  lie  the  foundations  of  the  practice  of  to-morrow. 

In  these  circumstances  it  appears  permissible  or  even  desirable 
to  enunciate  problems  the  solution  of  which  may  still  be  remote, 
although  in  the  attempt  contentious  hypotheses  must  figure 
as  prominently  as  facts  that  have  been  positively  ascertained. 
The  idea  of  the  South  Kensington  lectures  seems  to  admit  the 
possibility  of  the  written  versions  appearing  as  essays,  fugitive 
it  may  be,  but  at  the  time  serious,  and  justified  by  the  experi- 
ence that  the  impulse  to  honest  study,  the  most  that  a 
teacher  can  hope  to  impart,  comes  as  often  from  an  insight  into 
the  aims  that  direct  and  sustain  the  labours  of  others,  as  from 
the  most  lucid  exposition  and  the  most  judicious  selection  of 
accredited  dogmas. 

As  an  essay,  therefore,  these  pages  were  intended  to  point 
out  that  the  study  of  animal  metabolism  is  daily  becoming  less 


vi  PREFACE 

the  study  of  the  sum  total  of  chemical  change  in  the  body,  and 
more  the  study  of  the  individual  chemical  reactions,  the  items 
that  go  to  form  the  final  sum.  It  can  only  be  when  this 
tendency  has  worked  itself  out,  that  we  shall  know  how  far  the 
hopes  are  justified,  that  the  power  to  cope  with  disease  will  be 
increased  by  a  more  exact  knowledge  of  what  it  means  regarded 
as  a  chemical  problem.  At  present  it  is  difficult  to  form  any 
conception  of  the  future  progress  of  medicine,  to  which  the 
chemical  development  of  physiology  and  pathology  should  not 
contribute  more  than  it,  or  perhaps  any  other  development,  has 
contributed  in  the  past.  And  if  practical  medicine  appears  at 
the  present  time  to  owe  but  little  to  such  studies,  it  may  be  that 
the  glimpses  of  a  new  outlook  in  the  study  of  metabolism,  which 
are  yearly  becoming  more  frequent,  are  the  first  indications  of 
a  path  that  is  to  lead  us  far.  Disease  is  a  disturbance  in  the 
normal  balance  of  concurrent  chemical  changes,  and  the  balance 
is  in  the  first  instance  disturbed  because  some  one  or  other  of 
the  indispensable  chemical  reactions  is  either  precipitated  or 
checked.  It  is  difficult  to  believe  that  the  chances  of  our  being 
able  to  restore  the  balance  will  not  be  improved  the  more  we 
learn  of  the  nature  of  each  of  these  concurrent  changes.  To 
understand  the  structure  of  the  body,  it  has  been  necessary  to 
dissect  it ;'  to  understand  the  composite  whole  of  metabolism,  it 
must  be  analysed,  and  each  particular  reaction  studied  by  itself 
and  in  its  relations  to  the  whole.  This  is  beginning  to  be  done, 
and  that  is  why  there  are  many  who  look  forward  to  the  work 
of  the  coming  years  along  these  lines  with  hope  and  confidence. 


CONTENTS 

LECTURE  PAGE 

I.    INTRODUCTORY  :   PHYSIOLOGICAL  CHEMISTRY  AND  META- 
BOLISM .  .  .  .  .  ....        i 

II.  THE  ASSIMILATION  AND  SYNTHESIS  OF  CARBOHYDRATES.        20 

III.  THE  CATABOLISM  OF  CARBOHYDRATES          .  .  -5° 

IV.  THE  ASSIMILATION  AND  SYNTHESIS  OF  FAT          _,_         .        72 
V.  THE  CATABOLISM  OF  FAT        .                                            -97 

VI.    THE  ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  .  .  122 

VII.    THE  CATABOLISM  OF  PROTEIDS                     .           .  144 

VIII.    THE  METABOLISM  OF  CYCLIC  FORMATIONS  .  .  165 

INDEX                                                             .           •  •  *99 


vii 


ABBREVIATIONS 


Am.  Ch.  Jl.     .  American  Chemical  Journal, 
Am.  Jl.  Phys.  .     American  Journal  of  Physiology. 

Ann.        .        .  Annalen  der  Chemie  und  Pharmacie. 

A.  P.  I.    .        .  Annales  de  FInstitut  Pasteur. 

A.  St.  P.          .  Archives  des  Sciences  Biologiques  de  St  Peter sbourg. 

B.  .        .        .  Berichte  der  deutschen  Chemischen  Gesellschaft. 
B.  Cbl.     .        .  Biochemisches  Centralblatt. 

B.  k.  W.          .  Berliner  klinische  Wochenschrift. 
Cbl.f.k.M.     .  Centralblatt  filr  klinische  Medizin. 

C.  R.  S.  B.       .  Comptes  Rendus  de  la  Societe  Biologique. 

D.  A.  f.  k.  M.  Deutsches  Archiv  fiir  klinische  Medizin. 
D.  M.  IV.        .  Deutsche  Medizinische  Wochenschrift. 
D.-R.  A.          .  Dubois-Reymond1  s  Archiv  fur  Physiologie. 
Rrgeb.     .        .  Rrgebnisse  der  Physiologie. 

H.  B.  .  Hofmeister's    Beitrage    zur    Chemischen    Physiologie    und 

Pathologie. 

H.-S.  Z. .        .  Hoppe-SeyleSs  Zeitschrift fiir  Physiologische  Chemie. 

J.  C.  S.    .         .  Journal  of  the  Chemical  Society. 

Jl.  of  Phys.      .  Journal  of  Physiology. 

J.  Pr.  Ch.        .  Journal  fiir  Praktische  Chemie. 

M.f.  Ch.         .  Monatsheftefiir  Chemie. 

M.J.       .        .  Maly's  Jahresberichte  fiir  Thierchemie. 

M.  M.  W.       .  Miinchener  Medizinische  Wochenschrift. 

Pfl.  A.     .        .  Pfliiger's  Archiv  fiir  die  Gesammte  Physiologie. 

S.  A.       .        .  Schmiedeberg s  Archiv  fiir  Pharmakologie   und   Rxperi- 

mentelle  Pathologie. 

Sk.  A.  .  Skandinavisches  Archiv  fur  Physiologie. 

V.  A.       .        .  Virchow's    Archiv   fiir    Pathologie     und     Pathologische 

Anatomie. 

Z.f.B.    .        .  Zeitschrift  fiir  Biologie. 

Z.f.  k.  M.       .  Zeitschrift  fiir  klinische  Medizin. 


viii 


PROBLEMS    IN    ANIMAL 

METABOLISM  ||| 

LECTURE  I 

INTRODUCTORY:   PHYSIOLOGICAL  CHEMISTRY  AND 
METABOLISM 

METABOLISM  is  a  very  simple  phrase,  admirable  in  its  compre- 
hensiveness ;  it  covers  all  the  chemical  changes  in  living 
organisms  which  constitute  their  life,  the  changes  by  which  their 
food  is  assimilated  and  becomes  part  of  them,  the  changes 
which  it  undergoes  while  it  shares  their  life,  and  finally  those  by 
which  it  is  returned  to  the  condition  of  inanimate  matter. 
Gathered  together  under  this  one  phrase  are  some  of  the  most 
intricate  and  inaccessible  of  natural  phenomena.  It  implies  also, 
and  gently  insists  on  the  idea,  that  all  the  phenomena  of  life  are 
at  bottom  chemical  reactions.  When  a  muscle  twitches  no  less 
than  when  a  gland  secretes,  it  is  not  too  much  to  say  that  when 
we  are  moved  to  tears  or  laughter  it  is  chemical  reactions  that 
are  the  underlying  causes  to  which  ultimate  analysis  must  lead 
us.  When  it  is  possible  to  give  an  adequate  account  of  animal 
metabolism  in  this  sense,  it  is  clear  that  physiological  chemistry 
will  have  done  its  work  and  be  an  extinct  science.  But  it  is  not 
necessary  to  point  out  that  this  ideal  goal  lies  far  beyond  the 
horizon. 

And  yet  there  must  be  few  branches  of  science  in  which 
during  recent  decades  more  vigorous  activity  has  been  displayed 

A 


2  INTRODUCTORY  [LECT. 

or  in  which  what  has  been  achieved  holds  out  greater  promise 
for  the  near  future.  Especially  in  the  past  twenty  years  the 
number  and  the  energy  of  workers  in  physiological  chemistry 
has  increased  from  year  to  year,  and  the  progress  effected  by 
their  work  has  become  more  and  more  significant. 

At  the  same  time  it  is  remarkable  that  very  much  of  what  we 
value  most  in  the  contributions  made  during  this  period  to 
physiological  chemistry  has  not  been  immediately  concerned 
with  the  problems  of  metabolism.  It  is  rather  in  the  elucida- 
tion of  the  nature  and  constitution  of  the  material  in  which  the 
processes  of  life  are  carried  out,  than  in  the  interpretation  of  the 
chemical  changes  themselves,  that  progress  has  been  conspicuous. 
A  very  brief  reference  to  some  of  the  advances  made  during  the 
last  twenty-five  years,  which  are  and  always  will  be  landmarks 
in  the  history  of  this  branch  of  science,  will  suffice  to  illustrate 
this  fact,  and  at  the  same  time  show  how  it  is  that  while  the 
chemical  chapters  in  physiological  treatises  yearly  grow  in 
length,  the  chapters  on  metabolism  have  to  be  left  compara- 
tively little  changed. 

In  no  subject  is  the  progress  effected  greater  or  more 
familiar  than  in  the  chemistry  of  the  carbohydrates.  We  have 
learnt  that  the  simple  sugars  of  which  all  carbohydrates  are 
composed  are  either  aldehydes  or  ketones  of  polyatomic  alcohols, 
and  may  have  as  they  occur  in  nature  either  five  or  six  carbon 
atoms  in  chain :  that  they  have  therefore  three  or  four  unsym- 
metrical  carbon  atoms,  each  of  which,  that  is  to  say,  is  combined 
with  four  atoms  or  groups  of  atoms  that  are  all  different  from 
each  other :  that  consequently  the  different  possible  spatial 
arrangements  of  these  atoms  or  groups  of  atoms  about  each 
unsymmetrical  carbon  atom  give  rise  to  differences  between 
sugars  in  other  respects  identical :  galactose  and  glucose,  for 
instance,  are  both  aldehydes,  both  hexoses,  but  owing  to  the 
different  spatial  configuration  of  the  parts  of  the  molecule,  they 
are  endowed  with  properties  by  which  they  may  be  readily  dis- 
tinguished from  each  other.  The  comprehension  of  these 
fundamental  properties  of  the  sugars  has  led  to  the  determina- 
tion of  the  chemical  relationships  between  almost  all  the 


i.]  CHEMISTRY  OF  CARBOHYDRATES  3 

different  sugars  that  occur  in  living  organisms,  to  the  synthesis 
of  most  of  them,  and  the  conversion  of  many  of  them  in  vitro 
into  others.  The  work  principally  of  Emil  Fischer  has  taught 
us  exactly  where  in  the  molecule  each  atom  must  stand,  what  is 
formed  if  it  changes  places  with  one  of  the  other  groups  in  the 
molecule,  exactly  why  it  is  that  there  are  so  many  varieties  of 
sugar  in  nature  with  the  same  empirical  and  even  constitutional 
formula,  which  of  all  the  conceivable  dispositions  of  the  same 
atoms  and  groups  of  atoms  do  not  occur  in  nature,  and  how 
many  of  these  too  may  be  built  up.  The  chemistry  of  the 
carbohydrates  has  become  a  problem  in  geometrical  permuta- 
tions, and  almost  all  the  possible  permutations  have  been  identi- 
fied, and  most  of  these  synthesised.  And  at  the  same  time  we 
have  learnt  that  the  geometrical  character  of  chemical  problems 
so  conspicuous  among  the  carbohydrates  must  be  appreciated 
among  compounds  of  other  kinds  for  which  as  yet  we  have  no 
such  definite  data. 

No  less  remarkable  has  been  the  development  of  the 
chemistry  of  proteids.  The  very  first  things  we  learn  about 
proteids — the  colour  reactions — which  were  for  so  long  nothing 
but  empirical  tests,  are  now  rich  in  interest,  significance,  and 
associations.  In  the  xanthoproteic  reaction  we  recognise  the 
facility  with  which  aromatic  radicals  form  nitro-compounds, 
and  the  well-known  chromophoric  properties  of  — NO2  groups. 
Millon's  reaction,  which  is  a  general  reaction  for  mono-oxy- 
aromatic  compounds  provided  that  they  do  not  contain  — NO2 
groups,  reveals  the  presence  of  tyrosine.  But  far  more  interest- 
ing than  these  two  are  those  known  as  the  biuret  reaction,  and 
the  reaction  of  Adamkiewicz.  Many  other  substances  besides 
biuret  and  proteids  are  known  to  give  a  pink  tinge  to  alkaline 
solutions  of  copper.  H.  Schiff  studied  the  nature  of  these 
substances,  and  having  determined  their  common  characteristics, 
made  the  following  generalisation :  all  substances  that  contain 
two 


4  INTRODUCTORY  [LKCT. 

groups  united  by  the  carbon  atoms  either  directly  to  each  other 
or  to  the  same  carbon  or  nitrogen  atoms,  give  the  biuret  reaction. 
Oxamide, 


CO .  NH2 
CO .  NH0 


therefore,  and  malonamide, 


H2N 


CH2—  C/ 


behave  in  this  respect  similarly  to  biuret  itself,  which  is 

^C—  NH—  C^ 
H2N/  XNH2 

Inferences  as  to  the  constitution  of  proteid  molecules  were  indi- 
cated by  these  observations.  But  the  same  reaction  is  given  by 
a  basic  condensation  product  of  glycocoll,  amido-acetic  acid, 
which  was  first  described  many  years  ago  by  Curtius,  the  exact 
constitution  of  which  has  only  recently  been  determined.  And 
this  compound  is  not  covered  by  SchifFs  generalisation.  A 
number  of  other  condensation  products  of  glycocoll  and  other 
amido  acids,  to  which  Fischer  has  given  the  name  of  peptides, 
also  give  the  reaction.  The  general  formula  for  these  pep- 
tides  is  : 

H\  Q 

R\;c-c<  V 

H2N/  \N< 

X 

In  the  simplest  of  these  bodies,  glycylglycine,  the  value  of 
R  is  H,  and  of  Rj  is  —  CH2  .  COOH.  These  values  do  not  confer 
on  the  compound  the  property  of  giving  the  biuret  reaction  ;  but 
certain  other  values  do.  In  this  way  the  relationship  between 
these  compounds  on  the  one  hand,  and  oxamide  and  the  other 
substances  which  by  SchifFs  rule  give  the  biuret  reaction  on  the 
other  hand,  becomes  clear,  as  also  does  the  sort  of  expansion 
which  this  rule  must  undergo  in  order  to  include  the  poly- 


i.]  CHEMISTRY  OF  PROTKIDS  ft 

peptides  which  give  this  reaction.  In  the  expanding  process, 
however,  the  rule  loses  its  value ;  the  determining  conditions 
and  the  exceptions  become  too  complicated  to  be  included  in  a 
single  simple  sentence.  But  the  significance  of  what  we  have 
learnt  about  the  biuret  reaction  in  its  bearing  on  the  constitution 
of  proteid  molecules  has  become  very  great. 

For  these  molecules,  when  broken  up  by  the  hydrolytic 
action  of  acids  and  alkalies,  or  of  water  itself  at  high  tempera- 
tures or  in  the  presence  of  enzymes,  yield  large  quantities  of 
amido  acids,  and  must  therefore  be  very  largely  composed  of 
amido-acid  radicals.  And  under  these  conditions  the  polypep- 
tides  also  are  broken  up  into  their  constituent  amido  acids. 
It  is  fair  to  argue,  therefore,  that  proteids  and  polypeptides 
give  the  biuret  reaction,  because  they  both  contain  amido  acids 
combined  in  the  same  way.  Fischer's  polypeptides  which  give 
the  biuret  reaction  are  not  proteids,  but  they  are  very  closely 
related  to  that  large  part  of  all  proteid  molecules  to  which  the 
biuret  reaction  which  all  proteids  give  is  due.  The  first  step 
towards  the  synthesis  of  proteids  has  been  taken,  and  at  the 
same  time  the  meaning  of  the  most  universal  and  important 
of  all  the  proteid  reactions,  as  well  as  the  constitution  of  the 
greater  part  of  the  molecule  of  all  proteids,  has  been  elucidated. 

The  reaction  of  Adamkiewicz  also  has  an  interesting  story. 
For  a  long  time  it  was  reckoned  of  little  account ;  it  was  merely 
an  empirical  test,  a  trick  ;  and  not  reliable  as  that.  For,  carried 
out  according  to  the  original  prescriptions,  with  glacial  acetic 
acid  and  sulphuric  acid,  it  was  found  to  be  capricious,  and  fre- 
quently failed.  This  capriciousness  was  shown  by  Hopkins  to 
be  due  to  the  fact  that  glacial  acetic  acid  had  nothing  to  do  with 
the  reaction  :  when  glacial  acetic  acid  gave  the  test,  it  did  so 
because  it  contained  as  an  impurity  an  oxidation  product, 
glyoxylic  acid.  Glyoxylic  acid  is  not  always  present  in  acetic 
acid,  and  hence  the  uncertainty  of  the  test.  Carried  out  with 
glyoxylic  acid  itself,  the  reaction  is  exceedingly  delicate,  and 
quite  certain.  Hopkins,  moreover,  showed  what  it  was  in  the 
proteids  that  caused  them  to  give  the  reaction,  and  isolated 
as  a  crystalline  substance  a  new  component  of  the  proteid 


6  INTRODUCTORY  [LECT. 

molecule  which  proved  of  the  greatest  interest.  Not  only  is 
it  the  cause  of  the  Adamkiewicz  reaction,  it  is  the  substance 
produced  in  the  course  of  tryptic  digestion  of  proteids  which 
reacts  with  bromine  in  the  long-familiar  tryptophane  or  pro- 
teinochromogen  reaction ;  hence  the  name  tryptophane  given 
by  Hopkins  to  the  new  substance.  It  is  probably  the  one  and 
only  source  of  all  the  familiar  derivatives  of  indol  which  are 
formed  from  proteids  by  the  action  of  bacteria  in  the  intestine 
and  elsewhere,  and  therefore  of  the  indican  in  the  urine ;  it 
is  the  source  of  the  indigo  in  the  indigo  plant ;  and  since  it  is 
from  tryptophane  that  in  dogs  and  certain  other  animals,  a 
substance  found  in  the  urine,  kynurenic  acid,  which  is  a  quino- 
line  derivative,  is  formed,  it  has  even  been  suggested  that  it 
is  from  the  tryptophane  group  in  proteids  that  all  the  important 
vegetable  alkaloids  that  are  based  on  a  quinoline  or  pyridine 
ring  are  formed  in  the  course  of  the  proteid  metabolism  of  the 
plants  in  which  they  occur. 

As  soon,  therefore,  as  we  learn  anything  about  proteids  we 
learn  how  much  of  what  is  interesting  and  important  in  our 
knowledge  is  the  result  of  the  most  recent  work.  But  this  is 
of  course  only  a  small  part  of  what  the  period  under  considera- 
tion has  brought  to  light  on  this  subject.  The  study  of  the 
action  of  boiling  mineral  acids  on  proteids  was  begun  by  Liebig, 
and  in  his  laboratory  led  to  the  discovery  among  the  resulting 
products  of  leucine,  tyrosine,  glycocoll,  aspartic  and  glutamic 
acids.  But  as  the  first  simple  methods  of  separating  these 
products  from  the  mixtures  in  which  they  were  obtained  con- 
sisted in  direct  crystallisation  or  precipitation  with  metallic 
salts,  only  those  products  were  isolated  which  were  either 
particularly  abundant  or  particularly  insoluble.  And  the 
further  investigation  of  the  constitution  of  proteids  along  these 
lines  by  Schiitzenberger  and  others  led  to  little  that  was 
definite.  Within  the  last  twenty  years,  however,  on  two  occa- 
sions a  new  impulse  has  been  given  to  this  study,  which  has 
in  each  case  led  to  the  most  important  results. 

In  the  first  place,  the  use  of  phosphotungstic  acid  as  a  re- 
agent for  the  precipitation  of  organic  bases  led  to  Drechsel's 


i.]  CHEMISTRY  OF  PROTEIDS  7 

discovery  of  lysine  as  a  constituent  of  proteids,  and  this  to  the 
proof  given  by  Hedin  that  arginine  is  formed  in  considerable 
quantities  in  the  hydrolysis  of  most  proteids.  The  earliest 
fruitful  use  of  this  reagent  in  physiological  chemistry  was  in 
the  work  of  Schultze,  who  first  described  arginine  as  present 
in  young  seedlings ;  but  the  significance  of  this  discovery  was 
not  at  that  time  fully  understood.  Subsequently,  in  addition 
to  lysine  and  arginine  a  third  widely  distributed  basic  con- 
stituent of  proteids  was  added  to  the  list  by  Hedin  and  Kossel, 
who  independently  found  histidine  in  proteids  of  various  kinds. 

More  recently  still,  Emil  Fischer  has  applied  to  the  investi- 
gation of  the  cleavage  products  of  proteids  a  number  of  methods 
not  previously  made  use  of  for  this  purpose  ;  first  of  all  by 
converting  the  mono-amido  acids  into  their  ethyl  esters,  and 
distilling  these  in  vacua,  he  obtained  these  acids  in  a  pure  state, 
and  then  for  the  separation  of  the  different  components  of  each 
fraction  he  has  devised  new  methods  which  have  already  given 
excellent  results  ;  the  most  important  of  these  consist  in  the 
use  of  phenyl  isocyanate  and  naphthalene  sulphonyl  chloride. 
In  this  way  several  new  amido  acids  have  been  brought  to  light 
among  the  products  of  proteid  hydrolysis,  and  in  many  cases, 
too,  others  more  familiar  where  they  had  not  previously  been 
found.  Alanine,  phenyl-alanine,  amido-valerianic  acid,  serine, 
the  pyrrholidine  carboxylic  acids,  should  now  receive  recognition 
as  constituents  of  proteid  molecules  just  as  much  as  the  sub- 
stances discovered  by  Liebig  and  his  followers,  leucine,  tyrosine, 
glycocoll,  and  the  rest.  And  these  methods  for  isolating  the 
mono-amido  acids  are  likely  to  yield  still  further  results  of 
importance,  while  the  detailed  study  of  the  combinations  of 
the  amido  acids  which  is  being  carried  out  by  Fischer  is  not 
likely  to  stop  till  still  more  important  knowledge  of  the  con- 
stitution of  proteids  has  been  acquired. 

If  we  take  into  account  those  proteids  as  well  which  are 
of  a  more  complex  structure,  in  which  besides  a  typical  proteid 
component,  a  component  of  quite  a  different  nature  is  found, 
the  progress  that  has  been  made  in  the  comprehension  of  these 
prosthetic  groups,  as  they  have  been  called,  the  nucleic  acids^ 


8  INTRODUCTORY  [LECT. 

haematin,  and  the  carbohydrate  derivatives  which  combine  with 
proteids,  has  kept  pace  with  the  development  of  the  chemistry  of 
proteids  themselves.  Especially  in  the  case  of  the  nucleic  acids 
is  this  conspicuous.  Almost  all  that  is  known  of  these  sub- 
stances is  due  to  the  work  of  the  last  two  decades,  and  much  of 
this  is  so  fresh  and  familiar  that  it  is  sufficient  only  to  refer  to 
it.  In  the  chemistry  of  the  glyco-proteids,  the  first  and  still  the 
most  important  work  was  that  of  Schmiedeberg,  who  published 
his  account  of  the  carbohydrate  derivatives  obtained  from  carti- 
lage barely  fifteen  years  ago.  It  has  led  to  many  other  additions 
to  the  knowledge  of  this  class  of  compound  which  are  a  great 
advance  on  the  notions  current  twenty  years  ago.  And  what 
Schmiedeberg  did  for  the  glyco-proteids  Nencki  did  for 
hsematine ;  the  work  initiated  by  him  seems  likely  to  lead 
before  many  years  pass  to  the  synthesis  of  this  most 
important  pigment. 

Under  these  main  headings  and  a  few  minor  ones,  among 
which  the  determination  of  the  constitution  of  adrenaline  would 
find  a  place,  the  principal  achievements  in  what  has  been  the 
most  active  and  successful  period  in  the  history  of  physiological 
chemistry  would  probably  almost  all  be  included. 

It  is  not,  therefore,  the  difficulties  connected  with  metabolism 
that  have  been  removed.  It  is  rather  that  it  is  possible  now 
to  have  clearer  views  as  to  the  composition  and  constitution  of 
the  material  in  which  the  chemical  processes  of  life  are  carried 
out,  and  not  that  the  chemical  processes  themselves  are  clearer 
for  what  has  lately  been  accomplished  in  physiological  chemistry. 

The  knowledge  that  has  been  gained  may  be  looked  on  as 
a  sign  that  the  time  is  at  last  approaching  when  we  may  hope 
to  see  some  of  the  problems  solved.  To  set  about  the  study 
of  reactions  when  only  vague  ideas  about  the  nature  of  the 
reagents  are  possible,  is  not  likely  to  lead  to  the  desired  results. 
Much  of  the  discussion  of  problems  in  metabolism  must  have 
been  in  the  past,  and  must  even  now  be  premature  speculation. 
We  may  think  already  that  the  old  disputes  over  the  conver- 
sion of  proteids,  carbohydrates,  and  fats  into  each  other  were 
sometimes  entered  upon  with  little  more  regard  for  than  know- 


i.]  THE  CHEMICAL  POWERS  OF  CELLS  9 

ledge  of  the  chemical  nature  of  the  substances  dealt  with,  or 
of  the  character  of  the  changes  propounded ;  and  attempts  to 
handle  such  questions  now  are  still  not  likely  to  escape  a 
similar  judgment  in  the  future. 

Nevertheless  the  strictly  logical  order  has  been  departed 
from  in  some  investigations  that  have  led  to  valuable  results 
in  the  past,  and  we  still  must  wait  for  the  rational  elucidation 
of  facts  in  metabolism  that  were  proved  by  physiologists  long 
ago.  The  formation  of  fat  from  starch  by  animals  is  accepted 
as  proved,  no  less  than  the  conversion  of  sugar  into  carbonic 
acid  and  water.  Such  facts  we  are  accustomed  to  handle  with 
familiarity,  but  it  cannot  be  said  that  the  chemistry  of  either 
of  these  changes,  or  of  many  others  of  equally  fundamental 
importance,  is  much  better  understood  now  than  thirty  years 
ago.  In  the  subject  with  which  physiological  chemistry  deals 
there  is  much  that  has  been  built  up  solidly  and  substantially, 
as  a  wing  in  the  building  of  organic  chemistry;  and  it  is  the 
work  of  this  kind  that  has  raised  our  hope  in  recent  years. 
But  there  is  much  which  is  still  merely  of  the  nature  of 
scaffolding,  and  it  is  a  question  yet  whether  the  permanent 
structure  that  is  to  replace  this  scaffolding  can  be  built  of  the 
same  material  and  in  the  same  style  as  the  permanent  parts 
already  in  existence. 

It  has  often  been  pointed  out  that  no  laboratory  can  rival 
an  animal  cell.  Changes  that  we  know  take  place  in  living  cells 
can  often  be  effected  also  in  the  laboratory.  But  when  this  is 
the  case,  the  methods  of  the  laboratory  are  far  too  violent,  and 
generally  too  impotent  as  well,  for  it  to  be  possible  to  suppose 
that  they  reproduce  the  methods  by  which  the  living  cell  works. 
In  those  cases  in  which  it  is  sometimes  thought  that  the  steps  of 
a  reaction  carried  out  in  living  organisms  are  known,  these 
steps  may  be  laboriously  followed  in  the  laboratory,  but  only 
with  the  use  of  agencies,  and  at  a  cost  of  material,  which  show 
the  difference  between  the  conditions  of  the  two  procedures. 
Carbonic  acid  can  be  reduced  to  carbonic  oxide  by  passing  it 
over  red-hot  charcoal,  and  this  gas  acting  on  dry  potassium 
hydrate  is  converted  into  formic  acid.  Formate  of  lime  on  dry 


10  INTRODUCTORY  [LECT. 

distillation  yields  small  quantities  of  formic  aldehyde,  and  formic 
aldehyde  under  the  influence  of  lime-water  will  condense  to 
form  sugars,  among  which  are  small  quantities  of  a  sugar 
a-acrose,  from  which  the  sugars  that  occur  in  Nature  can  be 
prepared.  These  sugars  are  believed  to  be  formed  in  the  parts 
of  plants  which  contain  chlorophyll  from  carbonic  acid,  also  by 
way  of  carbonic  oxide  and  formic  aldehyde,  under  the  action  of 
sunlight  and  the  active  substances  that  exist  in  the  living  plant. 
But  if  the  stages  in  the  reaction  are  the  same  under  the  two  sets 
of  conditions,  there  is  nothing  else  that  is.  From  carbohydrates 
animals  certainly,  and  plants  probably,  make  fats  or  oils,  a 
transformation  at  which  we  can  only  stand  aghast ;  or  starch 
and  sugar  are  oxidised  smoothly  and  quietly  at  a  low  tempera- 
ture, at  which,  when  protected  from  contact  with  living  matter, 
they  show  no  tendency  whatever  to  oxidation.  The  compara- 
tively simple  changes  involved  in  condensation  and  hydrolysis 
present  just  as  striking  differences  between  the  methods  of  the 
laboratory  and  of  the  cell.  Hippuric  acid  is  very  readily 
obtained  by  allowing  benzoyl  chloride  to  act  on  amido-acetic 
acid  in  the  presence  of  caustic  alkali,  but  in  the  dog's  kidney  it 
is  made  no  less  easily,  without  the  use  of  either  the  acid 
chloride  or  caustic  alkali,  from  the  simple  acids  or  their  salts. 
Benzoic  acid  and  glycocoll  can,  it  is  true,  be  made  to  condense 
directly  in  vitro,  but  for  this  they  must  be  heated  together  in 
the  dry  state  in  a  sealed  tube  at  a  temperature  of  i6o°C.  for 
twelve  hours.  Urea  has  been  synthesised  artificially  from  the 
same  substances  as  those  from  which,  according  to  the  commonly 
accepted  theory  of  Schmiedeberg,  it  is  made  in  the  liver, 
ammonium  carbonate  or  carbamate.  But  to  prepare  it  from 
the  former  Drechsel  employed  either  the  alternating  current 
given  by  an  induction  coil  and  interrupter  worked  by  several 
Grove  cells,  or  a  constant  current  in  the  presence  of  platinum 
black  :  from  the  carbamate  he  obtained  it  by  heating  solutions 
in  sealed  tubes  to  I35°C.  And  in  every  case,  in  spite  of  the 
energetic  measures,  the  yield  was  small.  In  the  body  the  yield 
is  almost  quantitative,  and  the  conditions  are  the  ordinary 
normal  conditions  of  life.  For  the  hydrolytic  changes  brought 


i.]  THE  CHEMICAL  POWERS  OF  CELLS  11 

about  constantly  under  these  same  conditions  by  living  cells, 
we  have  to  use  high  temperatures  and  strong  acids  or  alkalies. 
Compare,  too,  the  procedure  by  which  taurine  has  lately  been 
obtained  from  cysteine  with  the  chemistry  of  the  cells.  The 
mercaptan  group  is  oxidised  to  the  sulphonic  acid  with  bromine, 
and  the  product  heated  with  water  in  a  sealed  tube  for  four 
hours  at  240°  C.  in  order  to  remove  carbonic  acid.  Another 
recent  instance  is  the  decomposition  of  arginine.  By  boiling 
this  substance  with  baryta  water,  Schultze  obtained  20  per  cent, 
of  the  theoretical  amount  of  urea,  and  Hedin,  in  the  same  way, 
from  20  g.  of  the  silver  salt  was  unable  to  get  enough 
urea  to  prepare  a  pure  specimen.  The  cells  of  the  liver  and 
other  organs,  acting  on  arginine,  make  a  clean  cleavage  into 
urea  and  ornithine :  in  one  experiment  Dakin  and  Kossel 
found  that  5  c.c.  of  the  fluid  expressed  from  crushed  liver  cells 
completely  converted  5  g.  of  arginine  into  its  components  in 
five  minutes.  Wherever  we  turn,  in  the  reactions  carried  out  in 
metabolism  agencies  appear  at  work  of  an  entirely  different 
character  from  those  of  which  we  have  command  in  the 
laboratory.  And  even  when  the  last  secrets  of  the  constitution 
of  proteids  have  been  laid  bare,  and  when  proteids  complete  in 
every  detail  have  been  synthesised,  this  supreme  achievement 
of  organic  chemistry  will  still  be  merely  introductory  to  the 
physiological  chemistry  which  is  to  give  us  an  insight  into  and 
teach  us  how  to  control  the  reactions  that  lie  behind  the 
phenomena  of  life. 

There  was  a  time  when  physiological  questions  were  debated 
without  regard  to  facts  that  could  be  ascertained  by  the  study 
of  anatomy.  This  was  succeeded  by  a  period  in  which  the 
study  of  physiology  was  temporarily  identified  with  the  study  of 
anatomy.  Then  when  all  that  anatomy  could  contribute  had 
been  learnt,  it  was  found  that  the  work  of  the  physiologist,  as 
we  now  understand  it,  was  only  beginning.  So,  too,  there  has 
been  a  time  when  the  problems  of  physiological  chemistry  have 
been  approached  with  but  little  knowledge  of  the  nature  of  its 
material  ;  a  period  when  this  branch  of  physiological  study 
began  to  appear  as  nothing  but  a  branch  of  organic  chemistry  : 


12  INTRODUCTORY  [LECT. 

but  sooner  or  later  it  must  be  recognised  that  there  is  work 
here  for  physiology  to  do  which  it  cannot  get  done  elsewhere, 
any  more  than  the  whole  of  physiology  could  be  learnt  by 
dissections. 

This  time  may  not  have  come.  There  is  certainly  very 
much  that  has  yet  to  be  learnt  that  is  of  the  nature  of  pure 
chemistry.  But  there  are  certain  conceptions  which  have  had 
a  growing  influence  in  recent  years,  and  which  promise  to  be 
of  great  significance  in  the  interpretation  of  physiological 
processes. 

Foremost  among  these  is  the  idea  of  the  part  played  in 
such  processes  by  enzymes.  In  1897,  Buchner  first  found  that 
a  substance  can  be  obtained  from  the  living  yeast  plant,  solu- 
tions of  which,  though  entirely  free  from  living  organisms  of 
any  kind,  cause  sugar  to  be  resolved  into  alcohol  and  carbonic 
acid,  just  as  the  living  yeast  cells  do.  The  inference  is  that, 
since  this  substance  is  found  in  the  plant,  when  the  plant 
ferments  sugar  it  does  so  because  it  contains  this  substance,  and 
not  because  it  is  alive.  The  difficulties  commented  on  above, 
of  tracing  any  similarity  between  the  conditions  under  which 
chemical  changes  are  effected  in  the  laboratory,  and  those 
under  which  the  same  changes  are  brought  about  in  living 
organisms,  led  to  the  assumption  that  life  was  a  cause  of 
chemical  change.  Vital  activity,  the  action  of  some  property 
of  living  things  which  is  not  to  be  defined  in  any  other  way, 
and  is  not  shared  by  anything  that  is  not  alive,  was  the  only 
explanation  forthcoming  to  meet  this  difficulty.  The  explana- 
tion was  merely  a  verbal  one,  but  there  was  no  other.  And  up 
to  the  time  of  Buchner's  discovery,  the  fermentation  of  sugar 
by  yeast  was  always  cited  as  the  typical  instance  of  fermenta- 
tion due  to  the  vital  activity  of  an  organism,  an  organised 
ferment ;  and  this  kind  of  fermentation  was  kept  strictly  distinct 
from  fermentations  brought  about  by  enzymes  or  unorganised 
ferments — chemical  substances,  that  is,  that  are  active  as  ferments 
where  there  is  no  life.  There  were  two  totally  distinct  causes 
of  fermentation  :  life,  and  enzymes.  When,  therefore,  what  had 
always  been  regarded  as  the  crucial  instance  of  the  ferment 


i.]  ENZYMES  AND  VITAL  ACTIVITY  13 

action  of  life  was  found  to  be  nothing  of  the  kind,  there  was  a 
movement  about  the  foundations  of  physiological  belief.  When 
yeast  ferments  sugar,  it  is  a  typical  enzyme  that  is  at  work ; 
the  only  difference  between  this  enzyme  and  the  typical 
"  unorganised  ferments  "  is  the  accidental  one  that  the  latter  are 
secreted  by  cells  so  as  to  act  in  external  media  removed  from 
the  cells,  whereas  this  acts  within  the  cells,  does  not  leave  them, 
and  is  therefore  not  to  be  found  elsewhere,  unless  special 
measures  are  taken  to  crush  the  cells  and  express  their  contents. 
The  surrender  of  this  point  need  not  involve,  and  has  not 
involved,  the  surrender  of  the  whole  position.  It  is  only  one 
among  myriads  of  changes  which  we  are  in  the  habit  of 
attributing  to  vital  activity.  But  it  has  made  more  obvious 
than  it  was  before,  that  in  regarding  life  as  the  cause  of  the 
chemical  reactions  underlying  the  phenomena  of  life,  we  are 
indulging  in  a  verbal  illusion.  It  has  called  to  mind,  too,  the 
fact  that  this  is  not  the  first  or  only  instance  in  which  important 
functions  in  metabolism  are  served  by  enzymes  that  are  not 
excreted  from  the  cells,  but  act  within  them.  More  than  forty 
years  before  this,  Claude  Bernard  had  ascribed  to  an  enzyme 
acting  in  the  liver  cells  the  conversion  of  stored  glycogen  into 
sugar  for  distribution  by  the  blood  to  other  parts  of  the  body. 
And  a  suggestion  made  about  the  same  time  by  Ludwig  has 
also  been  recalled.  Physiological  chemistry,  he  said,  may  some 
day  prove  to  be  a  chapter  in  the  chemistry  of  catalytic  action. 

It  is  remarkable  that  since  Buchner's  discovery,  and  in  part, 
no  doubt,  as  a  result  of  the  stimulus  it  gave,  a  large  number  of 
other  intracellular  enzymes  have  been  discovered,  so  that  our 
conception  of  the  use  made  of  enzymes  in  Nature  has  had  to  be 
very  considerably  extended.  The  first  enzymes  to  be  detected 
and  studied,  those  of  the  digestive  secretions,  form  a  class 
physiologically,  but  not  essentially,  distinct  from  the  others. 
They  are  discharged  from  the  cells,  and  serve  the  cells  by 
bringing  about  changes  in  the  fluid  which  is  in  contact  with  the 
cells.  The  ease  with  which  this  fluid  could  be  removed  from 
the  neighbourhood  of  the  cells  that  produce  it,  accounts  for  the 
fact  that  the  enzymes  of  this  class  were  for  so  long  the  only 


14  INTRODUCTORY  [LECT. 

substances  generally  recognised  as  having  the  properties  of 
enzymes,  and  also  for  the  fact  that  it  has  not  been  easy  to 
dissociate  the  idea  of  an  enzyme  from  that  of  a  secretion.  In 
addition  to  these  there  are  enzymes  which  are  to  be  found  only 
intimately  associated  with  the  cell  substance  and  confined 
within  the  limits  of  the  cell.  Among  these  there  are  some  the 
action  of  which  has  long  been  known,  because  the  products  of 
the  changes  they  set  up  are  formed  in  larger  quantities  than 
are  necessary  for  the  internal  economy  of  the  cells  themselves. 
Besides  the  enzyme  in  yeast  that  causes  alcoholic  fermentation, 
the  enzyme  that  causes  lactic  acid  fermentation,  in  the  Bacillus 
acidi  lactici,  has  been  isolated.1  The  other  fermentations  of  the 
same  class  which  have  not  been  proved  to  be  due  to  enzymes 
would  include,  to  name  the  most  familiar,  acetic  and  butyric 
fermentations  of  sugar  and  the  ammoniacal  fermentation  of 
urea.  In  the  sugar  fermentations  there  is  apparently  an 
extraordinary  extravagance  in  the  wanton  destruction  of  the 
food  stuff  of  the  cells.  It  is  partially  broken  down  by  them, 
and  the  products  are  turned  out  in  large  quantities  in  their 
environment.  It  is  common  to  speak  of  these  substances, 
alcohol  especially,  as  the  excreta  of  the  cells.  But  this  is  clearly 
incorrect.  In  the  formation  of  alcohol  from  sugar  only  4  per 
cent,  of  the  energy  of  the  sugar  is  liberated,  and  practically  none 
when  lactic  acid  is  formed.  These  substances  are  intermediate, 
not  final  products  of  metabolism.  The  formation  of  an  inter- 
mediate product  in  such  large  excess  over  the  needs  of  the 
organism  is  a  most  remarkable  phenomenon  in  the  economy  of 
the  species,  intelligible  conceivably  as  a  protective  device.  The 
over-production  of  a  special  substance,  to  which  the  species 
producing  it  is  less  sensitive  than  its  rivals  in  the  struggle  for 
existence,  might  serve  to  keep  these  rivals  away  from  the  feeding 
ground.  Milk  that  has  once  been  effectively  occupied  by  the 
lactic  acid  bacillus  is  a  preserve  in  which  poachers  do  not 
prosper. 

If  these  fermentations  are  all  of  them  the  work  of  intra- 

1  Herzog,  H.-S.  Z.  37,  381,  1903  ;  Buchner  and  Meisenheimer,  B.  36,  634, 
1903. 


i.]  INTRACELLULAR  ENZYMES  15 

cellular  enzymes,  as  is  known  to  be  the  case  in  the  instances  of 
alcoholic  and  lactic  acid  fermentation,  then  these  enzymes 
constitute  a  second  physiologically  distinct  group,  an  intra- 
cellular  group  characterised  by  the  over-production  of  a  meta- 
bolic product.1  The  other  intracellular  enzymes,  distinguished 
from  these  by  the  absence  of  this  characteristic,  have  been  the 
latest  to  draw  upon  themselves  the  attention  of  biologists.  Not 
only  are  they  confined  to  the  interior  of  the  cells,  but  the 
products  of  their  action  are  made  use  of  entirely  in  the  internal 
economy  of  the  cells ;  they  are  produced  in  quantities  that  can 
be  dealt  with  in  the  cell,  and  there  is  ordinarily  no  over- 
production. 

It  is  the  enzymes  of  this  physiological  group  that  are  of 
special  importance  in  the  interpretation  of  metabolic  processes. 
In  addition  to  hydrolytic  reactions  in  the  cells,  in  which  glycogen 
and  the  disaccharides,  fats  or  proteids,  are  resolved  into  their 
components,  there  is  a  great  variety  of  other  reactions  which 
have  been  ascribed  to  intracellular  enzymes.  The  oxidation  of 
aromatic  aldehydes,  the  oxidation  of  purine  bases,  the  oxidation 
of  tyrosine  ;  the  removal  of  amido  groups  from  amido  acids,  and 
of  urea  from  arginine  ;  the  decomposition  of  peroxides,  which  is 
sometimes  regarded  as  the  means  by  which  oxidation  changes 
are  restricted  to  the  appropriate  parts  of  the  body,  and  excluded, 
for  instance,  from  the  blood ;  the  decomposition  of  uric  acid,  of 
glucose,  and  of  other  substances  ;  all  these  and  many  more  have 
been  attributed  to  the  operation  of  enzymes  within  the  cells. 
It  is  very  possible  that  some  of  these  attributions  may  not  be 
confirmed.  But  the  extension  of  the  idea  of  enzyme  action  into 
all  phases  of  metabolic  activity  has  been  to  effect  a  revolution  in 
physiological  chemistry.  There  is  an  outlook  even  beyond  the 
limits,  to  which  it  is  hoped  that  organic  chemistry  may  carry  its 
study  of  the  composition  of  the  material  in  which  these  changes 
are  carried  out. 

The  question  rises  at  once :  what  is  the  precise  value  of  the 

1  In  such  a  group  would  be  included  the  enzyme  that  hydrolyses 
glycogen  in  the  liver,  and  others  familiar  in  the  physiology  of  mammalian 
animals. 


16  INTRODUCTORY  [LECT. 

new  conception  of  the  part  played  by  enzymes  in  animal  meta- 
bolism ?  Even  supposing  that  enzymes  are  to  account  for  all  that 
is  remarkable  in  biological  chemistry,  are  we  any  better  off  than 
when  it  was  all  accounted  for  by  the  vital  activities  of  the  cells  ? 
If  we  have  no  exact  idea  of  what  an  enzyme  is  and  how  it  works, 
it  may  appear  as  if  we  were  again  trying  to  satisfy  curiosity  with 
an  explanation  that  is  verbal  and  nothing  more.  It  is  true  that, 
though  the  attention  of  many  workers  in  many  countries  has 
been  concentrated  on  the  problems  of  enzyme  action  for  years, 
very  few  of  the  questions  on  this  subject  that  seem  most  urgent 
are  finally  settled.  But  a  very  suggestive  summarisation  of  the 
present  tendencies  is  contained  in  Ostwald's  definition  of  cata- 
lytic action,  which  was  intended  to  cover,  and  include  as  a  special 
instance  of  catalytic  action,  the  action  of  enzymes.  A  catalytic 
agent  is  a  substance  that  alters  the  velocity  with  which  a 
chemical  change  is  brought  about.  This  is  simplicity  itself: 
but  what  does  it  imply  ? 

A  change  in  the  velocity  of  reactions  can  also  be  brought 
about  by  a  change  in  the  temperature  to  which  the  reagents  are 
exposed.  It  has  been  found  that  every  rise  of  10°  in  the 
temperature  increases  the  velocity  of  most  reactions  from  two 
to  threefold,  which  means  that  a  reaction  which  at  o°  C.  lasts  a 
year  may  at  100°  C.  be  completed  in  less  than  seven  minutes. 
A  reaction  which  lasts  a  year,  we  are  apt  to  regard  as  one  that 
does  not  take  place.  If  a  catalytic  agent  effects  a  change  in  the 
velocity  of  a  reaction,  which  is  of  this  order  of  magnitude,  it  may 
appear  to  bring  about  a  reaction  that  does  not  otherwise  occur. 
We  say  that  pepsine  and  hydrochloric  acid  cause  proteolysis. 
But  proteids  are  hydrolysed  by  water  alone,  if  the  temperature 
is  high  enough,  at  160°  C.  for  instance.  If  the  rate  at  which  this 
hydrolysis  is  effected  at  i6o°C.  is  double  that  at  which  it  would 
be  effected  at  1 50°  C.  and  so  on  for  every  10°  lower,  then  in  six 
hours  at  160°  as  much  hydrolysis  would  be  attained  as  in  three 
years  at  40°  C.  But  in  six  hours  at  40°  C.,  pepsine  and  hydrochloric 
acid  can  accomplish  more  still,  so  in  this  case  the  acceleration  of 
hydrolysis  effected  by  the  enzyme  appears  to  be  greater  than 
that  due  to  a  rise  of  temperature  amounting  to  I2O°C. 


i.]  DEFINITION  OF  CATALYSIS  17 

If  enzymes  produce  changes  in  the  velocity  of  reactions  of 
this  order  of  magnitude,  then  the  fact  that  reactions  occur  in 
living  organisms  which  do  not  occur  in  vitro  may  have  to  be 
differently  stated ;  the  reactions  may  occur  in  vitro,  but  too 
slowly  to  be  detected. 

The  definition  implies,  therefore,  that  the  peculiarities  in  the 
behaviour  of  substances  in  living  organisms  where  enzymes  are 
at  work  may  be  due  not  to  the  endowment  of  matter  with 
properties  differing  in  kind  from  the  properties  it  has  in 
inanimate  forms ;  the  substances  do  not  react  differently,  but 
with  a  different  velocity.  The  general  properties  of  substances 
in  vitro  and  in  vivo  are  the  same,  and  the  general  laws  of 
reaction  in  vitro  are  applicable  to  reactions  in  vivoy  if  we  allow 
for  the  operation  of  catalysis.  And  it  is  well  known,  of  course, 
that  the  phenomena  of  catalysis  are  not  restricted  to  the 
products  of  life. 

But  it  also  implies  that  where  differences  in  the  nature  of 
reactions  appear  to  exist  in  comparing  animate  and  inanimate 
nature,  they  may  be  due  to  the  acceleration  of  one  line  of 
reaction  to  the  exclusion  of  others — a  selective  catalysis.  Yields 
in  the  laboratory  are  seldom  theoretical :  in  addition  to  the 
main  line  of  reaction  there  are  other  side  lines  or  branch  lines 
into  which  the  reaction  tends  to  be  diverted,  and  in  an  extreme 
case  there  may  be  no  detectable  yield  from  the  main  line  of 
reaction.  If  we  suppose  that  the  main  line  is  brought  under 
the  influence  of  a  catalytic  agent,  while  the  side  lines  are  not, 
then  the  yield  may  become  to  all  intents  theoretical.  Or  if  a 
side  line  is  so  influenced,  practically  the  whole  reaction  may  be 
guided  into  this  line  ;  and  if  the  side  reaction  is  one  that  in  vitro 
occurs  only  to  a  very  limited  extent,  so  that  it  altogether  escapes 
detection,  then  the  whole  course  of  chemical  change  may  appear 
to  be  altered  past  recognition,  and  a  reaction  of  which  in  vitro 
we  know  nothing  be  the  characteristic  feature  of  the  behaviour 
of  the  same  substances  in  the  presence  of  the  catalytic  agent. 
It  must  be  remembered,  too,  that  the  definition  includes  the  idea 
that  the  change  in  velocity  may  have  a  negative  sign,  and  that 
changes  that  we  are  in  the  habit  of  regarding  as  inevitable  may 

B 


18  INTRODUCTORY  [LECT. 

in    the    sphere   of    action    of   negative   catalysts    be    avoided 
altogether. 

This  conception  of  catalytic  action,  then,  suggests  that  much 
if  not  all  of  what  has  hitherto  appeared  to  separate  biological 
chemistry  from  the  chemistry  of  the  laboratory  may  ultimately 
disappear.  The  methods  of  physical  chemistry  may,  when 
developed  and  applied  to  biological  questions,  supply  what 
analytical  and  synthetic  methods  can  never  furnish. 

Something   more   than   this   may  be  implied  in  Ostwald's 
generalisation.     A  reaction  is  accelerated  by  a  rise  of  tempera- 
ture because  the  activity  of  molecular  movements  is  increased  as 
the  temperature  is  raised,  and  the  increased  activity  of  molecular 
movements  brings  about  more  frequent  encounters  of  the  react- 
ing substances.     The  more  frequently  the  reacting  molecules 
encounter  one  another,  the  greater  the  chance  of  their  reacting  on 
one  another.     But  the  frequency  with  which  such  encounters 
take  place  may  also  be  increased  by  increasing  the  number  of 
molecules  in  a  given  space.     If  two  solutions  completely  mixed 
react  with  a  certain  velocity  on  each  other  when  the  solution  is 
homogeneous,  of  equal  concentration  in  all  its  parts,  the  addition 
of  a  third  substance,  if  it  affects  the  homogeneity  of  the  solution, 
must  increase  the  concentration  of  dissolved  molecules  in  those 
parts  of  the  system  into  which  they  congregate.     The  velocity 
of  the  reaction  will  be  determined  by  the  concentration  of  the 
most  concentrated  portions  of  the  solution,  provided  the  rate  of 
diffusion  is  not  too  slow.     Solutions  in  which  fine  suspended 
particles  are  present  are  not  homogeneous.     Owing  to  adsorp- 
tion, the  concentration  of  dissolved  molecules  is  different  near  the 
surface  of  the  suspended  particles  from  what  it  is  elsewhere. 
Some  instances  of  catalytic  action  appear  to  be  due  to  the  dis- 
turbance   of    the    homogeneity    of    solutions    by    adsorption. 
Platinum  decomposes  hydrogen  peroxide,  and  the  amount  of 
hydrogen  peroxide  decomposed  varies,  not  with  the  mass  of  the 
platinum,  but  with  the  amount  of  platinum  surface.     Platinum 
black  produces  far  more  change  than  platinum  foil,  and  far  more 
active  than  platinum  black  are  the  so-called  colloidal  solutions 
of  platinum  obtained  by  setting   up  an  electric   arc  between 


i.]  ADSORPTION  AND  CATALYSIS  19 

platinum  electrodes  under  water.  If  the  catalytic  action  of 
platinum  is  due  to  the  peculiar  adsorptive  properties  of  platinum, 
as  Faraday  first  suggested,  that  of  other  catalytic  agents  may 
also  be  due  to  surface  properties ;  and  enzymes  in  particular,  in 
virtue  of  the  fact  that  they  are  colloidal  substances,  may  disturb 
the  homogeneity  of  solutions  in  which  they  are  suspended,  and 
owe  their  properties  in  part  to  the  alteration  of  the  concentration 
of  the  solution  in  the  neighbourhood  of  their  surfaces. 

Whether  the  whole  of  enzyme  action  is  to  be  accounted  for 
by  adsorption  phenomena,  it  is  not  necessary  for  our  argument 
in  this  connection  to  discuss.  There  are  other  conceptions 
which  physical  chemistry  is  at  present  making  use  of  in  order  to 
interpret  the  phenomena  of  catalysis  ;  but  in  these  conceptions, 
too,  surface  phenomena  play  a  large  part,  and  so  they  apply  to 
the  colloidal  enzymes  no  less  than  to  metallic  catalysts.  For 
our  present  purposes  it  is  sufficient  to  indicate  the  directions  in 
which  we  may  look  for  illumination  of  the  mysteries  of  meta- 
bolic processes.  Physical  chemistry  applied  to  biological 
problems  promises  to  supplement  the  analytical  and  synthetic 
work  of  the  past  twenty-five  years,  and  to  do  as  much  for  the 
elucidation  of  those  problems  as  organic  chemistry  has  done  and 
is  doing  to  determine  the  nature  of  the  material  with  which  in 
the  physiology  of  metabolism  we  are  concerned.  Physical 
chemistry  is  engaged  in  the  study  of  the  nature  of  colloidal 
solutions  and  their  properties,  and  also  in  that  of  the  phenomena 
of  catalysis  and  their  interpretation.  Till  we  have  learnt  all 
that  can  be  taught  us  by  the  results  of  this  study,  it  is  undesir- 
able and  unnecessary  that  we  should  hopelessly  resign  ourselves 
to  the  belief  that 'the  puzzles  of  animal  metabolism  must  for  ever 
remain  unsolved. 


LECTURE  II 

THE   ASSIMILATION   AND  SYNTHESIS  OF   CARBOHYDRATES 

ONE  of  the  most  interesting  of  all  the  chemical  reactions  that 
have  been  studied  in  biology  is  that  by  which  the  synthesis  of 
carbohydrates  is  carried  out  in  plants  from  the  carbon  dioxide 
in  the  air.  In  this  synthesis,  which  is  the  work  of  the  pigment 
chlorophyll,  the  first  stage  must  necessarily  be  a  reduction. 
The  suggestion,  made  by  Bayer  many  years  ago,  that  formic 
aldehyde  was  the  product  formed  in  this  reduction  process,  and 
that  the  carbohydrate  synthesis  took  place  by  condensation  of 
a  number  of  molecules  of  formic  aldehyde  into  one  large 
molecule,  has  never  been  completely  proved  to  be  correct.  But 
the  condensation  which  the  theory  supposes  to  take  place  is  a 
general  reaction  with  aldehydes,  known  as  the  aldol  condensation, 
from  the  product  of  condensation  of  two  molecules  of  acetic 
aldehyde,  aldol  — 

,Q  /OH  /O 

2  CH3  .  Cf     =   CH3  .  C<       .    CH2  .  Cf 

>H  NH  \H 

And  it  is  well  known  that  substances  with  the  constitution  and 
properties  of  sugar  have  actually  been  synthesised  in  vitro  from 
formic  aldehyde  by  the  help  of  the  condensing  action  of  alkalies. 
Six  molecules  of  formic  aldehyde  condensing  by  aldol  con- 
densation — 

,0  /OH       /OH       /OH       /OH       /OH      /O 

6H.cf     .   H.C<       .  C         .    C         .    C         .    C         .    C 

X  \ 


H 


—  would  clearly  produce  a  hexose  sugar. 

20 


LECT.  ii  ]         SYNTHESIS  OF  SUGAR  IN  ANIMALS  21 

That  the  carbohydrates,  free  or  combined,  found  in  animals 
are  ever  formed  by  such  a  fundamental  synthesis  as  this,  has 
never  been  contended.  Sugar  and  compounds  such  as  starch, 
from  which  sugar  is  easily  formed  by  changes  that  are  among 
the  most  familiar  in  physiological  chemistry,  are  so  abundant  in 
the  food  of  animals,  that  the  bulk  at  any  rate  of  carbohydrates 
found  in  animals  may  certainly  be  traced  to  the  substances  of 
this  nature  taken  into  the  body  ready-made  in  the  food.  But  it 
should  be  remembered  that,  if  the  course  of  the  reaction  by 
which  plants  synthesise  carbohydrate  from  the  carbonic  acid  of 
the  air  really  corresponds  to  the  scheme  propounded  by  Bayer, 
the  two  stages  in  this  synthesis,  the  reduction  and  the  condensa- 
tion, are  of  very  different  character  and  significance.  Aldol 
condensation  is  an  exothermic,  almost  an  isothermic  reaction — 

2  g.  mol  Acetic  aldehyde  \ /  I  g.  mol.  Aldol 

=  551  cal.  /  I  =  546.8  cal. 

But  extrinsic  sources  of  energy  are  necessary  for  the  conversion 
of  carbonic  acid  into  formic  aldehyde,  and  that  stage  of  the 
reaction  is  from  all  points  of  view  a  most  exceptional  one.  But, 
given  the  formic  aldehyde,  all  that  follows  is  comparatively 
comprehensible  and  commonplace.  And  if  in  animal  physiology 
we  are  driven  to  the  conclusion  that  a  true  synthesis  of  carbo- 
hydrate does  occur  in  the  animal  body,  there  is  no  peculiar 
difficulty  in  supposing  that  it  might  occur  by  the  same  kind  of 
condensation  as  Bayer  has  hypothesised  for  the  synthesis  of 
starch  in  plants. 

The  carbohydrate  which  is  found  free  and  uncombined  in 
the  animal  body  is  in  one  or  other  of  three  forms :  glucose, 
lactose,  glycogen.  Glucose,  the  most  widely  distributed  of  all 
the  sugars  in  Nature,  we  may  for  the  moment  suppose  to  be 
provided  in  sufficient  quantity  in  the  food,  either  as  free  glucose, 
or  in  the  form  of  disaccharides  or  starch,  which  in  digestion  are 
hydrolysed,  and  so  give  rise  to  glucose.  But  glycogen,  a 
polymeric  anhydride  of  glucose  found  only  in  animals,  must  be 
synthetically  produced,  even  in  such  animals  as  take  only 
animal  food,  since  even  the  glycogen  taken  as  food  is  hydrolysed 


22  ASSIMILATION  OF  CARBOHYDRATES  [LECT. 

in  digestion.  It  is  well  known  that  animals  fed  on  glucose  or 
starch  are  found  to  contain  much  more  glycogen  than  animals 
that  are  given  no  carbohydrate  in  their  food,  and  the  condensa- 
tion of  glucose  to  form  glycogen  is  the  most  familiar  of  all  the 
carbohydrate  syntheses  that  take  place  in  the  animal  body,  and 
one  that  is  admitted  on  all  hands.  The  strict  proof  of  its 
occurrence  was  furnished  by  the  experiments  of  Otto,  in  Voit's 
laboratory,  on  fowls  and  rabbits.  A  rabbit  was  kept  without 
food  for  four  days,  then  given  80  g.  of  glucose,  and  eight  hours 
later  killed.  In  the  liver  alone  more  than  9  g.  of  glycogen 
was  found,  or  nearly  17  per  cent,  of  the  whole  weight  of  the 
liver,  and  nearly  as  much  more  in  the  rest  of  the  body.  The 
largest  amount  of  glycogen  that  has  been  recorded  in  the  liver 
of  rabbits  starved  for  a  similar  period  is  less  than  half  a  gramme 
for  animals  of  the  weight  of  the  one  in  Otto's  experiment.1 
The  liver  alone,  therefore,  in  this  case  contained  nearly  9  g  of 
glycogen  more  than  could  be  expected,  unless  the  animal  could 
make  glycogen  from  glucose.  And  that  the  glycogen  was  not 
derived  from  proteid,  rendered  available  for  this  purpose  by  the 
large  supply  of  sugar,  was  quite  clear ;  for  the  nitrogen  excreted 
during  the  experiment  corresponded  only  to  about  5  g.  of  proteid, 
and  it  is  impossible  to  obtain  from  5  g.  of  proteid  9  g.  of 
glycogen  under  any  hypothesis.2 

How  this  synthesis  is  brought  about  we  do  not  know.  The 
fact  that  the  reversed  change,  from  glycogen  to  glucose,  is 
carried  out  by  an  enzyme  or  enzymes  obtained  from  the  same 
organ,  the  liver,  suggests  that  it  may  be  a  case  of  reversible 
enzyme  action.  The  enzymes  that  hydrolyse  glycogen  which 
are  found  in  the  saliva  and  pancreatic  juice  take  the  hydrolysis 
as  far  only  as  maltose,  and  the  conversion  of  maltose  into 
glucose  is  effected  in  digestion  by  a  different  enzyme,  maltase. 
But  the  two  stages  into  which  the  conversion  of  glycogen  into 
glucose  can  under  these  conditions  be  resolved  involve,  so  far  as 
we  know,  very  closely  related  reactions,  and  one  of  these,  the 
action  of  maltase,  is  of  all  enzyme  actions  the  one  in  which 

1  Kiilz,  Beitrage  zur  Kenntniss  des  Glycogens,  Marburg,  1891. 

2  Voit,  Z.f.  B.  28,245,  1892. 


ii.]  FORMATION  OF  GLYCOGEN  23 

reversibility  has  been  most  completely  established.1  If  this 
reversibility  of  enzyme  action  is  to  be  extended  to  the  action  of 
other  enzymes  than  those  for  which  it  has  been  proved,  then  one 
of  the  first  reactions  to  be  included  under  such  a  generalisation 
would  naturally  be  that  with  which  we  are  now  concerned.  The 
enzyme  that  hydrolyses  glycogen,  we  must  in  that  case  suppose, 
does  not  do  so  completely.  Its  action  comes  to  a  stop  at  the 
equilibrium  point,  when  a  certain  small  portion  of  the  glycogen 
is  left  unattacked.  If  less  than  this  small  amount  is  present 
within  the  sphere  of  action  of  the  enzyme,  then  some  of  the 
glucose  must  be  converted  into  glycogen.  And  if  the  glycogen 
as  soon  as  it  is  formed  is  removed  from  the  sphere  of  action  of 
the  enzyme,  either  by  entering  at  once  into  chemical  combina- 
tions, in  which  it  is  no  longer  subject  to  the  operation  of  the 
enzyme,  or  in  any  other  way,  then  so  long  as  this  is  the  case  the 
enzyme  would  continue  to  produce  glycogen,  and  no  glycogen 
would  be  hydrolysed.  As  soon  as  the  glycogen,  on  the  other 
hand,  was  set  at  liberty  again  and  permitted  to  come  under  the 
influence  of  the  enzyme,  the  opposite  change  would  be  brought 
about,  glucose  would  be  formed,  and  the  more  rapidly  the 
glucose  was  carried  away  by  the  blood,  or  otherwise  placed  out 
of  reach  of  the  enzyme,  the  more  rapidly  would  fresh  glucose 
be  formed.  Knowing  as  we  do  that  not  only  is  glycogen  formed 
from  glucose  in  the  liver,  but  glucose  is  also  formed  from 
glycogen,  and  that  this  latter  is  the  work  of  an  enzyme  that  can 
be  demonstrated  in  an  extract  of  the  organ,  it  is  specially 
tempting  to  imagine  that  this  is  an  instance  of  reversible 
zymolysis  ;  but  we  have  no  direct  evidence  that  it  is  so. 

In  this  synthesis  of  glycogen,  which  consists  in  the  formation 
of  a  polymeric  anhydride  of  glucose,  it  is  difficult  to  see  how 
anything  can  take  the  place  of  the  glucose,  unless  it  is  by  being 
first  converted  into  glucose.  If,  therefore,  we  have  reason  to 
believe  that  other  substances  of  any  kind  besides  glucose  can 
give  rise  in  the  body  to  the  formation  of  glycogen,  the  pre- 
sumption is  that  they  do  so  because  they  can  be  converted  into 
glucose.  We  may  leave,  therefore,  the  question  of  the  formation 
1  A.  Croft  Hill,/  C.  S.  73,  634,  1898. 


24  ASSIMILATION  OF  CARBOHYDRATES  [LECT. 

of  glycogen  from  substances  other  than  glucose,  till  we  are  led 
back  to  it  again  by  the  consideration  of  the  substances  from 
which  glucose  can  be  derived. 

The  other  carbohydrate  occurring  free  and  uncombined  in 
the  animal  body  is  lactose.  This  is  a  disaccharide  differing  from 
maltose  in  that  it  is  compounded,  not  of  two  molecules  of 
glucose,  but  of  a  glucose  molecule  and  a  molecule  of  galactose. 
The  difference  between  galactose  and  glucose  is  a  stereochemical 
one,  consisting  in  the  different  spatial  configurations  of  certain 
parts  of  the  molecule.  The  formulae  by  which  these  differences 
are  represented  are : 

OH  H       OH      OH 

COH .  C     .  C     .     C  .     C     .     CH2OH     for  Glucose 

H  OH       H         H 
and 

OH  H         H       OH 

COH.C     .  C     .     C  .     C     .     CH2OH     for  Galactose. 

H  OH      OH       H 

If  the  carbon  atoms  in  these  formulae  are  denoted  by  the 
numbers  I  to  6,  beginning  with  the  carbon  atom  in  the  aldehyde 
group  at  the  left-hand  end,  as  given  here,  then  the  difference 
between  galactose  and  glucose  consists  only  in  the  transposition 
of  the  hydrogen  and  hydroxyl  groups  connected  with  the  fourth 
carbon  atom. 

Now  if  galactose  as  well  as  glucose  is  present  in  the  food 
of  animals  during  the  secretion  of  milk,  it  is  possible  that  the 
synthesis  of  lactose  is  merely  another  case  of  the  condensation 
of  sugars  to  form  anhydrides,  very  similar  to  the  condensation 
of  glucose  to  form  glycogen.  Galactose  is  fairly  widely  dis- 
tributed in  nature.  It  occurs  principally  in  the  form  of  poly- 
saccharides,  compounded  of  galactose  and  a  pentose,  either 
arabinose  or  xylose.  Galacto-arabane  is  found  in  the  seeds  of 
beans,  peas,  vetches,  and  cresses,  and  the  young  plants  of  clover 
and  lucerne :  galacto-xylane  is  found  in  the  cereals  wheat  and 
barley.  It  is  possible,  therefore,  that  a  part  of  the  galactose 
in  the  sugar  of  milk  is  derived  without  change  from  the 
galactose  in  the  food  of  some  animals.  A  cow  yielding  2  to  3 


ii.]  ORIGIN  OF  GALACTOSE  25 

gallons,  20  to  30  Ibs.  of  milk,  turns  out  daily  about  I  Ib.  of 
sugar,  half  of  which  is  galactose.  It  is  not  known  to  what 
extent  the  galacto-pentanes  are  digested,  and  the  galactose 
set  free  from  them  absorbed.  But  it  is  not  necessary  to 
suppose  that  any  of  the  galactose  in  the  sugar  of  milk  is 
derived  directly  from  this  same  sugar  in  the  food.  For  it  is 
known  that  carnivorous  animals  secrete  undiminished  quantities 
of  lactose  when  kept  on  a  pure  meat  diet,1  so  that  the  power 
of  making  galactose  must  be  present  at  least  in  these  animals  : 
and  it  is  not  likely  to  be  confined  to  them.  It  is  most  natural 
to  suppose  that  this  galactose  is  formed  from  glucose  by  the 
transposition  of  a  hydrogen  atom  and  hydroxl  group,  as  indicated 
in  the  stereochemical  structural  formulae  given  above  —  such 
isomeric  transformations  among  the  sugars  were  the  subject 
of  the  important  studies  of  Lobry  de  Bruyn  and  van  Ekenstein.2 
They  showed  that  glucose  exposed  to  the  action  of  bases  in 
weak  solutions  gradually  lost  its  rotatory  power  :  for  instance, 
if  20  g.  of  glucose  in  500  c.c.  of  water  containing  10  c.c.  of 
normal  potash  was  heated  to  63°  C.  for  2\  hours,  the  rotation 
sank  from  +  5°  30'  to  ±  ic/:  and  they  proved  that  this  change 
was  due  to  the  conversion  of  part  of  the  glucose  into  fructose 
(laevulose)  and  mannose,  both  of  which  they  isolated  and 
identified.  Now  the  differences  between  these  three  sugars 
are  confined  to  the  first  two  carbon  atoms  in  the  formulae, 
written  as  above  for  glucose  and  galactose,  thus  : 
OH  ,H 

COH.C        .      C  =   Glucose. 


COH.C        .      C  =   Mannose. 

\OH 


CH2OH  .  CO  .  C  =   Fructose  or  Lsevulose. 


NDH 


1  Szubotin,  V.  A.,  p.  561,  1866. 

2  Recueil  des  travaux  chimiques  des  Pays  Das^  xiv,  and  xvi.,  1895  and  1897. 


26  ASSIMILATION  OF  CARBOFIYDRATES  [LECT, 

The  Dutch  chemists  originally  suggested  that  the  inter- 
changeability  of  these  three  forms  of  sugar  could  be  best 
explained  by  supposing  that  the  aldehyde  group  took  the 
hydrated  form,  and  from  this  a  hypothetical  anhydride  was 
formed  which  was  a  link  between  glucose  and  fructose,  thus  : 

/OH  .OH 

(1)  HO.C         —      C  —         =   Glucose  hydrate. 

\H  \H 

/°\ 

(2)  HO  .  C     —     C  =  hypothetical  Anhydride 

\TT         \w  °f  glucose  hydrate. 

/H  11  ' 

(3)  HO.C        —CO     —  =  Fructose. 


These  changes  being  reversible,  there  would  tend  to  be 
formed  from  fructose  (3),  in  addition  to  the  anhydride  (2), 
another  stereoisomeric  anhydride,  in  which  the  oxygen  atom 
linking  the  two  carbon  atoms  took  up  position  on  the  other 
side  of  the  molecule,  thus  : 

/H       H 

(4)     HO  .  C     —     C  =  hypothetical  Anhydride 

of  mannose  hydrate. 


/H     /H 

(5)     HO .  C        .      C  =  Mannose  hydrate. 

\3H      \3H 

And  so  fructose  could  be  converted  into  either  mannose  or 
glucose,  and  glucose  and  mannose  into  each  other  through 
fructose. 

The  same  chemists,  however,  subsequently  found  that  in 
addition  to  fructose  other  ketone  sugars  are  formed  at  the 
same  time,  and  for  these  the  theory  they  had  given  did  not 
provide.  But  it  does  not  follow  that  the  theory  is  untenable 


II.] 


ISOMERIC  TRANSFORMATIONS 


27 


for  the  changes  in  which  fructose  is  concerned.  There  is  reason 
for  thinking  that  glucose  also  tends  to  assume  the  form  in 
which  the  first  and  fourth  carbon  atoms  are  united  to  the 
same  oxygen  atom  instead  of  the  first  and  second,  as  supposed 
in  the  theory  of  Lobry  de  Bruyn  (formula  (2)  supra).  The 
methyl  glucoside  formed  by  heating  glucose  with  methyl  alcohol 
in  the  presence  of  hydrochloric  acid  has  lost  the  power  of 
reducing  copper  oxide,  and  therefore  has  no  longer  the 
aldehyde  group,  and  occurs  in  two  stereoisomeric  forms,  owing 
to  the  asymmetric  properties  acquired  by  the  carbon  atom 
of  the  aldehyde  group.  The  formula  ascribed  to  it  by  Fischer 
is: 

O 


(CH3 . 0  or)  H 


(H  or)  O  .  CH 


H 


It  is  clear  that  such  a  formula  for  glucose  derivatives  suggests 
that  glucose  and  galactose  may  be  convertible  into  each  other 
by  a  reaction  similar  to  the  one  proposed  by  Lobry  de  Bruyn, 
to  explain  the  conversion  of  mannose  into  glucose.  It  is  true 
that  this  chemist  expressly  investigated  this  question,  and  found 
that  potash  does  not  give  rise  to  the  formation  of  galactose 
from  glucose.  But  in  such  points  the  action  of  different  bases 
may  well  differ,  just  as  the  change  from  mannonic  acid  to 
gluconic  acid  is  not  effected  by  inorganic  bases,  but  is  by 
pyridine  and  quinoline.  At  any  rate,  in  the  animal  body  the 
isomeric  transformation  of  glucose  into  galactose  seems  to 
occur,  unless  we  prefer  to  think  that  the  galactose  is  derived 
from  substances  less  closely  related  to  it ;  and  if  it  occurs,  the 
problem  as  to  how  it  is  brought  about  has  to  be  solved. 

And  this  is  not  the  only  case  of  isomeric  transformation 
among  the  sugars  which  there  is  reason  for  believing  to  be 
brought  about  in  animal  chemistry.  Laevulose  certainly,  and 
mannose  almost  certainly,  as  well  as  probably  galactose  itself, 


28  ASSIMILATION  OF  CARBOHYDRATES  [LECT. 

have  been  shown  to  give  rise  to  the  formation  of  glycogen  in 
the  liver  in  the  same  way  as  glucose.  It  is  also  known  that 
the  glycogen  so  formed  is  the  same  glycogen  that  is  formed 
from  glucose,  so  that  it  is  clear  that  the  glycogen  cannot  be 
formed  till  the  transformation  of  these  sugars  into  glucose  has 
taken  place. 

In  addition  to  the  carbohydrates  that  occur  free  in  the 
body,  there  are  others  that  are  found  in  combination  with 
other  groups  of  different  kinds.  The  best  known  of  these  are 
the  carbohydrate  combinations  in  nucleic  acids,  in  certain  fatty 
acid  compounds,  and  in  certain  proteids.  In  these  cases, 
though  it  is  true  little  is  known  as  to  the  exact  nature  and 
constitution  of  these  compounds,  the  nature  of  the  sugar  has 
been  determined,  and  in  each  instance  presents  points  of 
interest. 

Every  addition  to  what  is  known  of  the  chemical  nature 
and  properties  of  nucleic  acids  must  be  of  importance.  Nucleic 
acids  and  protamines  together  make  up  almost  the  whole  of 
the  functionally  essential  part  of  the  spermatozoa.  Nucleic 
acids  in  combination  with  some  form  of  proteid,  in  the  nucleus 
of  every  cell,  may  fairly  be  looked  to  as  the  key  to  the 
mysteries  of  the  chemistry  of  living  matter.  Not  the  least 
interesting  of  the  many  striking  facts  that  have  in  recent  years 
been  elicited  with  regard  to  their  nature  and  constitution,  is 
that  some  form  of  carbohydrate  is  always  to  be  recognised  in 
their  molecules.  In  all  those  of  animal  origin  in  which  the 
nature  of  this  carbohydrate  has  been  determined,  it  has  proved 
to  be  a  pentose.  Hammarsten  was  the  first  to  suspect  the 
presence  of  a  five-carbon  sugar  in  a  nucleo-proteid ; 1  he  was 
able  to  isolate,  in  the  form  of  a  syrup,  a  substance,  obtained 
by  boiling  the  nucleo-proteids  of  the  pancreas  with  weak 
sulphuric  acid,  and  this  syrup  gave  reactions  characteristic  of 
pentoses.  The  osazone  melted  at  about  150°  C,  the  substance 
itself  gave  the  phloroglucine  reaction,  and  on  distillation  with 
hydrochloric  acid,  furfurol  was  found  in  the  distillate.  But 
since  these  reactions  might  have  been  accounted  for  by 
1  Hammarsten,  If.-S.  Z.  19,  27,  1893, 


ii.]  ORIGIN  OF  NUCLEIN  PENTOSE  29 

glycuronic  acid,  he  was  unable  to  say  positively  that  the 
substance  obtained  was  a  pentose.  Salkowski  proved  that  it  was, 
by  analysing  the  osazone.1  It  was  then  shown  by  Bang  that 
the  pentose  was  a  component  of  the  nucleic  acid,  and  not  of 
the  proteid  part  of  the  molecule.2  And  the  exact  nature  of 
the  sugar  was  determined  by  Neuberg,3  who  showed  that  it 
was  /-xylose,  the  sugar,  that  is,  with  the  constitution  represented 
by  the  formula — 

OH         H  OH 

COH.C C C CH2OH 

\  \  \ 

H          OH  H 

A  pentose  has  been  obtained,  too,  from  the  nucleic  acids 
found  in  the  liver,  kidney,  spleen,  thymus,  thyroid,  submaxillary 
gland,  brain,  and  muscle ;  and  in  the  case  of  that  from  the  liver, 
it  has  been  proved  that  it  too  is  /-xylose.4  In  the  nucleic  acid 
from  the  lymph  glands  Bang  was  unable  to  find  a  pentose.5 

Pentoses  of  all  kinds  appear  to  be  but  very  imperfectly 
assimilated  when  taken  with  the  food.  Xylose  itself  under 
these  conditions  is  excreted  unchanged  in  the  urine  in  amounts 
up  to  55  per  cent,  of  that  taken;  the  xylose,  therefore,  in  the 
nucleic  acids  is  in  all  probability  formed  in  the  body.  From 
the  formulae  for  xylose  and  glucose  it  is  clear  that  the  structure 
of  the  two  sugars  is  identical  as  far  as  the  fourth  carbon  atom, 
and  that  the  formula  of  xylose  may  be  obtained  by  leaving  out 
the  fifth  carbon  atom  in  the  formula  for  glucose.  Xylose  can 
be  obtained  from  glucose :  by  oxidation  of  the  terminal 
secondary  alcohol  group  in  glucose,  glycuronic  acid  is  formed ; 
and  Salkowski  and  Neuberg  showed  that  by  the  action  of 
certain  bacteria  on  glycuronic  acid  carbonic  acid  is  split  off 
and  the  sugar  /-xylose  formed.6  Now  it  is  almost  certain  that 

1  Salkowski,  B.  k.   W.,  No.  17,  p.  361,  1895  ;  and,  H.-S.  Z.  27,  p.  537, 
1899. 

2  Bang,  H.-S.  Z,  26,  145,  1898.  3  Neuberg,  B.  35,  1467,  1902. 
4  Wohlgemuth,  H.-S.  Z.  37,  475,  1903.           5  Bang,  H.  B.  4,  124,  1903. 

6  Salkowski  and  Neuberg,  H.-S.  Z.  36,  261,  1902. 


30  ASSIMILATION  OF  CARBOHYDRATES  [LECT. 

glycuronic  acid  is  formed  in  the  animal  body  from  glucose.  It 
is  known  that  camphor  is  excreted  in  the  urine,  combined  with 
glycuronic  acid.  P.  Mayer  found  that  a  rabbit,  when  in  addition 
to  its  ordinary  food  it  was  given  2  g.  of  camphor,  excreted 
2.18  g.  of  glycuronic  acid;  if  then  kept  without  food  for  nine 
days,  so  as  to  reduce  the  stock  of  carbohydrate  in  its  body,  the 
same  dose  of  camphor  caused  only  1.2  g.  of  glycuronic  acid  to 
appear  in  the  urine :  two  days  later  the  same  animal,  still  with- 
out food,  when  given  together  with  the  same  dose  of  camphor 
10  g.  of  glucose,  then  excreted  2.06  g.  of  the  acid.1  This  points 
clearly  to  glucose  being  the  source  from  which  glycuronic  acid 
is  formed,  in  the  rabbit  at  any  rate.  In  small  amounts,  glycuronic 
acid  is  found  constantly  in  normal  urine.  If,  therefore,  the 
reaction  by  which  glycuronic  acid  gives  up  carbonic  acid  and 
becomes  xylose  is  a  general  reaction  in  living  organisms,  and 
not  a  peculiarity  of  bacterial  chemistry,  it  is  possible  to  account 
for  the  xylose  necessary  for  the  synthesis  of  nucleic  acids  by  the 
metabolism  of  glucose.2 

The  exact  constitution  of  nucleic  acids  and  the  relationship 
of  the  pentose  to  all  the  different  parts  of  the  molecule,  is  not 
known.  But  the  nucleic  acids  do  not  reduce,  so  that  they  are 
of  the  nature  of  glucosides,  and  the  aldehyde  group  is  effaced 
in  combining,  probably  with  an  alcoholic  hydroxyl  in  the 
glycerine  phosphoric  acid.  This  union  is  not  dissolved  by 
trypsine,  but  in  the  combined  action  of  the  enzymes  of  the 
pancreatic  cells,  in  autolysis  of  the  pancreas,  the  pentose  is  set 
free,  and  can  be  separated,  as  the  phenylosazone.3 

The  combinations  in  which  carbohydrates  are  found 
associated  with  higher  fatty  acids  are  in  part  at  any  rate  like 
the  nucleic  acids,  in  that  their  exact  constitution  has  not  yet 
been  determined,  although  the  nature  of  the  sugar  has  been 
determined.  Cerebrines,  or  cerebrosides  as  they  have  been 
called,  to  indicate  the  presence  of  a  sugar  in  their  composition, 

1  P.  Mayer,  Z.f.  k.  M.  47,  68,  1902. 

2  Cf.  Salkowski  and  Neuberg,  loc.  cit. 

3  Cf.  Neuberg  and  Milchner,  B.  k.  W.,  p.  1081,  Oct.  10,  1904  ;  cf.  also, 
Bang,  H.-S.  Z.  31,  425,  1900  ;  and,  Burian,  Ergeb.  3,  87,  1904  ;  and,  B.  37, 696. 


ii.]  COMPOUNDS  OF  FAT  AND  SUGAR  31 

have  been  obtained  in  a  crystalline  form  from  the  brain.  The 
sugar  that  is  set  free  on  hydrolysis  of  these  cerebrines  has  been 
shown  to  be  galactose,1  while  the  rest  of  the  molecule  is  probably 
composed  of  higher  fatty  acid  radicals,  or  simple  nitrogenous 
derivatives  of  these.2  In  addition  to  the  cerebrine  compounds 
of  galactose,  higher  fatty  acids  compounded  with  sugar  were 
found  in  the  substance  jecorine,  which  Drechsel  first  obtained 
from  the  liver,  and  others  after  him  in  the  blood  and  elsewhere. 
The  nature  of  this  too  has  not  yet  been  exactly  defined. 
Drechsel 3  described  it  as  a  compound  of  sugar  with  lecithine  and 
some  other  group  or  groups,  in  which  sulphur  was  present,  and 
characterised  it  by  its  intensely  hygroscopic  properties,  its 
solubility  in  moist  ether,  and  insolubility  in  alcohol.  Combina- 
tions of  lecithine  with  sugar  have  been  described  as  jecorine 
by  Bing  and  others,  who  have  not  exactly  followed  Drechsel's 
methods  of  preparation.4  But  beyond  the  fact  that  lecithine 
may  cause  sugar  to  dissolve  in  ether,  and  sugar  may  cause 
lecithine  to  dissolve  in  aqueous  fluids,  such  as  the  blood,  and 
the  fact  that  such  combinations  of  sugar  and  lecithine  are 
to  be  found  in  the  yolk  of  eggs,  and  in  the  blood  and  certain 
organs,  nothing  definite  is  known  of  them.  But  jecorine  and 
the  cerebines  alike  serve  to  show  the  complexity  of  the  relation- 
ships into  which  the  simple  sugars  may  enter  in  the  animal 
body.  Mayer,  who  has  shown  that  glycuronic  acid  is  normally 
present  in  the  blood,  is  inclined  to  think  that  jecorine  is  a 
compound  of  glycuronic  acid  and  lecithine ;  but  this  has  not 
yet  been  demonstrated. 

With  regard  to  the  combinations  into  which  carbohydrates 
enter  with  proteids,  there  is  a  great  deal  that  is  still  not  clear. 
But  two  facts  at  any  rate  stand  out  from  much  that  is  obscure  : 
in  the  first  place,  that  the  number  of  proteids  into  the  composi- 
tion of  which  recognisable  derivatives  of  sugar  enter  are  much 
more  numerous  than  was  generally  believed  a  few  years  ago ; 

1  Thierfelder,  H.-S.  Z.  14,  209,  1890.  2  Bethe,  S.  A.  48,  73,  1902. 

3  Drechsel,  Ber.  der  Sachs. ges.  der  Wissensch.,  1866,  p.  44  ;  and,/.  Pr.  Ch. 

33,  425. 

4  Bing,  Sk.  A.  9,  336,  1899  ;  cf.,  too,  Mayer,  H.-S.  Z.  32,  530,  1901. 


32  ASSIMILATION  OF  CARBOHYDRATES  [LECT. 

and,  secondly,  that  in  a  majority  of  cases  at  any  rate  the  sugar 
has  been  found  to  be  present  in  the  form  of  glucosamine. 

It  has  long  been  known  that  a  certain  class  of  proteids  when 
boiled  with  weak  mineral  acids  give  solutions  that  reduce 
Fehling's  solution.  The  reduction  is  due  to  the  presence  of 
some  substance  closely  related  to  sugar  ;  for  from  the  solution 
a  phenylosazone  can  be  obtained.  Proteids  of  this  class  were 
known,  therefore,  as  the  glyco-proteids,  mucin  being  the  most 
familiar  of  the  class.  From  mucin  and  the  mucoid  proteids  of 
egg-white  and  of  ovarian  tumours  glucosamine  has  been 
definitely  isolated.  But  in  addition  to  these  glyco-proteids 
many  other  proteids,  not  previously  reckoned  in  this  class, 
have  been  suspected  of  containing  sugar  radicals,  and  in  some 
cases  proved  to  contain  glucosamine.  So  typical  a  "  native 
albumin "  as  egg-albumin  was  the  first  to  be  shown  to  share 
this  property  with  the  glyco-proteids.  The  proteid  of  the  yolk 
of  eggs  has  also  been  found  to  contain  glucosamine,1  and  so 
too  crystalline  serum-albumin,  from  human  as  well  as  from 
horses'  blood,2  and  serum-globulin.3 

The  amount  of  glucosamine  that  can  be  obtained  from  these 
different  proteids  is  often  very  small,  but  in  some  cases  is  very 
considerable ;  in  the  case  of  mucin  and  ovomucoid,  more  than 
30  per  cent,  but  in  serum-globulin  not  more  than  2  per  cent, 
and  still  less  in  serum-albumin,  while  egg-albumin  takes 
an  intermediate  position  with  10,  or  perhaps  nearer  15,  per 
cent.4 

Glucosamine  has  been  shown  by  Fischer  to  be  really  an 
amido-glucose,  so  that  the  old  name  chitosamine  is  to  be 
dropped.  The  constitution  is  expressed  in  the  formula — 


,H  /OH  /OH 

OH 


.    c/      . 
\H 


COH.CH.NH2.C<          .     C<          .     C<         .     CH2OH 


1  Neuberg,  B.  34,  3963,  1901.  2  Langstein,  H.  B.  i,  259,  1902. 

3  Langstein,  Krgeb.  3,  463,  1904. 

4  Hofmeister,  H.-S.  Z.  24,  159;  Langstein,  Ergeb.  i.,  94,  and  iii.,  463  ; 
and,  H.-S.  Z.  31,  491,  1900. 


ii.]  SUGAR  RADICALS  IN  PROTEIDS  33 

the  exact  disposition  of  the  groups  connected  with  the  second 
carbon  atom  being  as  yet  undetermined.1 

Evidence  of  the  presence  of  sugar  radicals  in  proteids,  less 
convincing  but  easier  to  obtain  than  that  given  by  the  isolation 
and  identification  of  glucosamine,  or  its  oxidation  product 
norisosaccharic  acid,2  is  afforded  by  certain  colour  reactions. 
The  purple  colour  given  by  sugar  with  a-naphthol  and  sul- 
phuric acid,  due  to  the  formation  of  furfurol  (Molisch's  reaction), 
is  also  given  by  a  large  number  of  proteids,  and  the  isolation 
of  sugar  derivatives  from  some  of  these  has  led  to  the  inference 
that  all  proteids  which  give  this  reaction  have  a  sugar  group 
in  their  molecules.  The  test  is  extremely  delicate ;  it  is  given 
by  as  little  as  0.5  mg.  of  cellulose,  for  instance,  so  that  its  very 
sensitiveness  raises  suspicions  as  to  the  legitimacy  of  such  an 
inference.  But  even  so  there  are  some  proteids  that  do  not 
even  by  this  test  show  signs  of  a  carbohydrate  group,  notably 
casein  and  gelatine. 

Another  reaction  recommended  for  the  testing  for  sugar 
groups  in  proteid  molecules  is  the  modified  orcine  test  of  Bial.3 
The  fluid  to  be  tested  is  heated  with  two  volumes  of  a  solution 
of  orcine  in  strong  hydrochloric  acid,  as  in  the  test  for  pentoses 
or  glycuronic  acid ;  only  a  drop  of  ferric  chloride  solution  is 
added.  Dextrose  and  galactose,  or  disaccharides  into  which 
these  sugars  are  compounded,  give  a  bluish-green  precipitate. 
A  solution  of  this  precipitate  in  amyl-alcohol  gives  a  character- 
istic spectrum,  in  which  the  yellow  and  the  edge  of  the  green 
is  blotted  out.  Laevulose  and  glucosamine  do  not  give  the 
reaction.  But  certain  proteids  do;  egg-albumin  and  the 
proteid  of  the  yolk  of  eggs,  serum-albumin  and  serum-globulin, 
but  not  casein  or  pseudo-mucin.  Since  glucosamine  does  not 
react  in  this  way,  it  is  argued,  there  must  be  another  carbo- 
hydrate group  in  addition  to  glucosamine  in  these  proteids. 
And  in  some  instances  carbohydrate  derivatives  more  complex 
than  glucosamine  have  been  obtained  from  proteids.  Frankel 

1  Fischer  and  Leuchs,  J3.  36,  24,  1903. 

2  Neuberg  and  Wolff,  B.  34,  3840,  1901. 

3  Bial,  Z.J.  k.  M.  50,  417  ;  M.  /.,  102,  1903. 


34  ASSIMILATION  OF  CARBOHYDRATES  [LECT. 

described  a  compound  carbohydrate  containing  nitrogen  which 
he  prepared  from  egg-albumin,  and  took  for  a  diamino-disac- 
charide ; l  and  Langstein  has  obtained  a  polyhexosamine  from 
serum-globulin  in  small  quantities.2  Chondrosine  was  the  first 
of  all  the  compound  carbohydrate  derivatives  to  be  obtained 
from  any  proteid,  and  Schmiedeberg  ascribed  to  it  the  con- 
stitution of  a  compound  of  glucosamine  with  glycuronic  acid, 
coupled  by  condensation  of  the  aldehyde  group  of  the  latter 
with  the  amine  group  of  the  former.  Orgler  and  Neuberg  have 
prepared,  also  from  cartilage,  a  substance  of  much  higher  mole- 
cular weight  than  corresponds  to  this  formula,  from  which  they 
failed  to  obtain  either  glucosamine  or  glycuronic  acid.  They 
found  instead  a  tetra-oxyamido-caproic  acid  and  some  carbo- 
hydrate substance,  the  nature  of  which  they  did  not  determine.3 
Besides  these  there  are  certain  compounds  of  the  nature  of 
albumoses,  into  the  composition  of  which  sugar  enters,  that 
have  been  described  by  Pick  4  and  by  Simon.5 

It  has  been  very  commonly  assumed  that  these  sugar  groups 
in  proteid  molecules  are  built  up  into  the  molecules  as  in  gluco- 
sides.  As  we  have  seen,  Neuberg  and  Milchner6  think  that  this 
mode  of  union  holds  for  the  pentose  in  nucleic  acid,  but  they 
argue  for  a  different  one  for  the  glucosamine  in  proteids.  The 
formation  of  a  glucoside  requires  a  hydroxyl  or  sulph-hydro 
group  in  an  organic  compound,  in  addition  to  the  sugar.  Of 
such  groups  in  proteids,  the  only  ones  known  are  in  tyrosine, 
cysteine,  oxyproline,  and  certain  other  oxyamido  acids.  These 
oxy-acids  are,  however,  present  only  in  very  small  amounts: 
cysteine  does  not  occur  as  such,  but  as  cystine,  in  which  the 
— SH  group  no  longer  exists  ;  and  since  the  glyco-proteids  give 
Millon's  reaction,  the  phenol  group  in  tyrosine  cannot  be  used 
for  glucoside  formation.  On  these  and  other  grounds,  they 

1  S.  Frankel,  M.f.  Ch.,  19,  819  ;  M.J.,  p.  23,  1898. 

2  L.  Langstein,  M.  f.  Ch,  24,  445  ;  M.  /.,  p.  30,  1903  ;  and,  Ergeb.  iii., 
460,  1904  ;  cf,,  too,  Leathes,  S.  A.  43,  245,  1899. 

3  Orgler  and  Neuberg,  H.-S.  Z.  37,  407,  1903. 

4  Pick,  H.  JB.  ii.,  481,  1902.  5  Simon,  S.  A.  49,  457,  1903. 

6  Neuberg  and  Milchner,  B.  k.  W.,  p.  1081,  Oct.  10,  1904 ;  cf.  sup.  p.  30. 


ii.]  SUGAR  SYNTHESIS  IN  GLYCOSURIA  35 

think  that  it  must  be  through  the  — NH2  group  that  glucosamine 
is  linked  on  to  the  proteid  molecule. 

It  is  clear  that  there  remains  still  very  much  to  be  learnt 
concerning  the  combinations  into  which  glucosamine  or  other- 
carbohydrate  derivatives  enter  with  proteids,  and  the  manner 
in  which  the  union  is  effected.  If  it  is  true  that  serum-albumin 
contains  less  than  I  per  cent,  of  glucosamine,  since  glucosamine 
has  the  molecular  weight  179,  the  molecular  weight  of  the 
albumin  comes  out  very  high,  over  18,000;  and  if  the  carbo- 
hydrate in  the  globulin  of  blood  is,  as  Langstein  thinks,  a  poly- 
hexosamine,  and  there  is  less  than  I  per  cent,  of  it,1  the 
molecular  weight  for  globulin  must  come  out  considerably 
higher  still,  something  like  50,000.  On  the  other  hand,  mucin 
containing  35  per  cent,  must  either  have  a  low  molecular 
weight,  or  else  present  a  very  considerable  number  of  groups 
into  which  the  glucosamine  can  be  welded. 

There  are  special  reasons  why  it  is  important  that  we  should 
get  clearer  conceptions  as  to  the  significance  and  prevalence 
of  carbohydrate  radicals  in  proteids.  In  the  ordinary  conditions 
of  animal,  and  particularly  of  human  life,  starch  and  sugar  enter 
so  largely  into  the  food  on  which  that  life  is  sustained,  that 
it  may  seem  unwarrantable  to  suppose  that  the  carbohydrates 
of  the  body  are  derived  from  anything  but  the  carbohydrates 
of  the  food.  And  yet  the  evidence  is  overwhelming  that  sugar 
can  be  formed  in  our  bodies  from  substances  which  are  not  in 
the  least  related  in  the  ordinary  chemical  sense  to  the  sugars. 
It  is  well  known  that  the  stock  of  carbohydrate  in  the  body 
is  never  large.  Animals  have  a  large  amount  of  proteid  in 
their  blood  and  tissues,  much  of  which  can,  if  necessary,  be 
made  use  of  as  a  source  of  energy,  both  heat  and  work.  The 
5  or  6  litres  of  blood  in  a  man's  body  contain  about  18  to 
20  per  cent,  of  proteids,  or  about  a  kilogramme  in  all.  The 
muscles  contain  about  20  per  cent,  of  proteids ;  and  since 
they  make  up  some  40  per  cent,  of  the  whole  body-weight, 
their  proteids  amount  to  8  per  cent,  of  it,  or  nearly  6  kilo- 
grammes. So  too  with  the  fats,  though  they  constitute  a  very 
1  Langstein,  M.f.  Ch.  26,  531,  1905. 


36  SYNTHESIS  OF  CARBOHYDRATES  [LECT. 

variable  fraction  of  the  whole  weight  of  the  body,  nevertheless 
in  normally  nourished  individuals  they  are  hardly  less  abundant 
than  the  proteids.  A  moderately  fat  dog  was  found  to  have 
26  per  cent,  of  fat ; l  and  as  much  as  45  per  cent,  has  been 
recorded  in  a  dog.  But  with  the  carbohydrates  the  figures  are 
very  different  from  these.  The  sugar  that  can  be  detected  in 
the  blood  in  health  at  the  most  amounts  to  0.2  per  cent,  or 
in  all  some  10  or  12  g.  In  the  liver  it  is  unusual  to  find 
more  than  10  per  cent,  of  glycogen,  and  more  than  20  per  cent, 
has  not  been  ever  observed ;  in  all,  therefore,  there  may  be 
about  200  to  400  g.  at  the  outside  in  the  liver.  In  the  muscles 
0.5  to  I  per  cent,  is  probably  not  too  low  a  figure  at  any  rate, 
and  that  would  give  another  150  to  300  g.  Exact  determina- 
tions of  the  amount  of  glycogen  in  the  human  body  have  not 
been  made,  but  it  is  seldom  likely  to  be  more  than  a  kilo- 
gramme. In  seven  dogs,  specially  fed  up  in  Pfliiger's  laboratory 
in  order  to  determine  the  maximum  amount  of  glycogen  that 
may  occur  in  these  animals,  the  average  reckoned  on  the  body- 
weight  was  about  2  per  cent,  but  in  one  case  nearly  4  per 
cent.  These  are  certainly  extreme  figures.  And  yet  it  is  on 
record  that  a  diabetic  patient  on  a  strictly  limited  diet  excreted 
no  less  than  1150  g.  of  sugar  daily.2  And  clinical  observers 
throughout  the  world  are  in  agreement  on  this,  that  in  the 
worst  cases  of  diabetes,  kept  on  the  strictest  diet,  whatever  it 
is  that  gives  rise  to  the  sugar  that  is  excreted  it  cannot  be 
only  the  carbohydrates  of  the  body.  Sugar  must  be  synthe- 
sised  under  such  circumstances,  often  on  a  very  large  scale. 
Experimental  pathology  confirms  in  this  the  conclusions  of  the 
clinical  pathologists.  Most  of  those  who  have  studied  the 
glycosuria  set  up  by  the  administration  of  phlorrhizine  have 
been  led  to  the  belief  that  the  sugar  is  often  excreted  in  such 
quantities  that  it  cannot  be  accounted  for  by  the  carbohydrate 
of  the  body.  And  in  the  glycosuria  following  removal  of  the 
pancreas  in  dogs  it  has  been  proved  irrefutably  that  this  is  so. 
Liithje  kept  a  dog  weighing  18  kg.  without  food  for  five  days, 

1  Mockel,  Pfl.  A.  1 08,  189,  1905. 

2  Niccolini,  B.  Cbl.  ii.,  p.  229,  1904. 


ii.]  PROTEIDS  AS  A  SOURCE  OF  GLYCOGEN  37 

and  then  excised  its  pancreas.  During  the  next  fourteen  days 
the  dog,  still  without  food,  excreted  228  g.  of  sugar ;  for  the 
ten  subsequent  days  it  had  a  diet  consisting  solely  of  proteid, 
a  preparation  of  caseine,  nutrose,  free  from  carbohydrate  of  any 
kind,  and  during  that  time  excreted  975  g.  of  sugar.  After 
this,  with  no  food  at  all  for  a  week,  it  excreted  1 50  g.  more. 
Altogether,  therefore,  it  excreted  1350  g.  of  sugar  without 
having  any  sugar  provided,  and  as  the  experiment  did  not 
begin  till  the  animal  had  been  without  food  for  five  days,  its 
body  must  have  been  but  poorly  stocked  with  glycogen  to  start 
with.  This  quantity  of  sugar,  amounting  to  7.5  per  cent,  of 
the  animal's  weight,  is  nearly  twice  as  great  as  the  highest 
amount  of  carbohydrate  ever  found  under  exceptional  circum- 
stances in  a  dog,  namely,  4  per  cent,  of  the  body-weight ;  so 
that  at  least  one-half  of  it  must  certainly  have  been  made  by 
synthetic  processes  in  the  body.1  Pfliiger  excised  the  pancreas 
from  a  dog  weighing  at  first  12  kg.  In  two  months,  3097  g.  of 
sugar  were  passed  in  the  urine,  whilst  the  dog's  food  contained 
no  carbohydrate  in  any  form,  and  practically  nothing  but  pro- 
teid. Synthetic  processes  must  in  any  case  have  produced  as 
much  as  2.5  kg.  of  sugar  in  this  experiment.2 

It  is  not  only  the  sugar  excreted  in  disease  that  may  be  the 
product  of  synthetic  changes  set  up  in  other  substances  in  the 
animal  body.  The  glycogen  of  the  liver  and  of  the  muscles  is 
also  most  probably  in  part  synthesised  from  proteid  ;  this  was 
first  taught  by  Cl.  Bernard,  and  subsequently  by  Kiilz,  Naunyn, 
v.  Mering,  and  others,  who  took  greater  precautions  to  eliminate 
sources  of  error.  It  is  true  that  the  results  of  none  of  these 
experiments  are  as  conclusive  as  those  referred  to  above  on  the 
excretion  of  sugar.  In  the  first  place,  the  glycogen  in  an 
animal's  liver  cannot  be  determined  more  than  once.  In  order, 
therefore,  to  decide  whether  there  is  more  glycogen  than  there 
would  have  been  if  the  treatment  had  been  different,  estimations 
have  to  be  made  in  control  animals.  But  animals  treated  and 
fed  in  exactly  the  same  way,  however  similar  they  may  be,  are 

1  Liithje,  D.  A.  /.  k.  M.  79,  1904. 

2  Pfliiger,  Pfl.  A.  108,  115,  1905. 


38  SYNTHESIS  OF  CARBOHYDRATES  [LECT. 

found  to  have  very  widely  different  amounts  of  glycogen  in 
their  livers.  So  that  the  conclusions  drawn  from  control 
animals,  though  they  may  be  probably  correct  when  a  sufficient 
number  of  controls  is  taken,  cannot  be  regarded  even  then  as 
positively  certain.  Neither  is  it  possible  to  be  certain  that  any 
particular  period  of  starvation  will  cause  the  glycogen  com- 
pletely to  disappear ;  so  that  if,  after  a  period  of  starvation 
followed  by  feeding  with  any  particular  food,  it  is  found  that 
glycogen  is  present  in  the  liver,  it  is  impossible  to  say  that  the 
glycogen  has  been  formed  from  that  food  :  it  may  be  probable, 
but  it  cannot  be  certain.  Pfliiger  found  in  a  dog  that  weighed 
33  kg.  nearly  50  g.  of  glycogen  after  starving  for  twenty-eight 
days,  and  there  was  nearly  5  per  cent  of  glycogen  in  its  liver. 
Such  a  case  is  exceptional,  but  it  must  be  taken  into  account. 

But  whether  we  are  convinced  by  the  experiments  which 
Kiilz  and  others  have  advanced  in  proof  of  the  formation  of 
hepatic  glycogen  from  proteid  food,  or  whether  with  Pfliiger  we 
insist  on  proof  of  this,  from  which  there  is  no  imaginable  loophole 
of  escape,  and  refuse  absolutely  to  take  account  of  probabilities, 
the  fact  is  established  that  the  synthesis  of  sugar  can  be  carried 
out  in  the  animal  body  from  something  which  is  not  sugar.  It 
has  been  established  in  diabetes  and  in  the  glycosuria  following 
excision  of  the  pancreas  in  dogs.  It  is  therefore  one  of  the 
chemical  reactions  of  animal  metabolism,  and  it  is  improbable 
that  this  reaction  is  peculiar  to  the  pathological  conditions  in 
which  it  has  been  possible  to  prove  it. 

The  material  used  for  this  synthesis  must  be  derived  either 
from  proteid  or  from  fat.  Some  of  the  results  referred  to  above 
seem  to  point  clearly  to  proteid  as  the  source  of  this  material. 
Luthje's  dog,  that  after  removal  of  its  pancreas  excreted  228  g. 
of  sugar  in  the  first  fourteen  days,  during  which  it  had  no  food, 
excreted  975  g.  in  the  next  ten  days,  when  it  had  a  proteid  diet 
free  from  carbohydrate  and  fat ;  that  is,  a  daily  average  output  of 
sugar  eight  times  as  great  as  before.  In  many  cases  of  diabetes, 
it  has  been  pointed  out  repeatedly,  the  output  of  sugar  varies 
with  the  amount  of  proteid  in  the  food.  The  experiments  of 
Bernard,  Kiilz,  Naunyn,  and  others,  on  the  formation  of  glycogen 


ii.]  THE  RATIO  D:N  39 

from  proteid  food  may  be  inconclusive  in  the  strict  sense,  but 
they  establish  some  degree  of  probability  that  proteid  can 
normally  furnish  material  for  carbohydrate  synthesis.  Min- 
kowski  tried  to  identify  the  source  of  the  sugar  by  seeing  if 
there  was  any  proportionality  between  the  amount  of  sugar 
excreted  and  the  amount  of  proteid  broken  down  in  the  body, 
the  nitrogen  of  which  was  cast  off  in  the  urine.  From  100  g.  of 
proteid  containing  17  g.  of  nitrogen,  urea  containing  about  7  g. 
of  carbon  would  be  formed.  The  rest  of  the  carbon  in  the 
proteid,  not  accounted  for  in  the  urea,  would  amount  to  about 
45  g.  Glucose  contains  40  per  cent,  of  carbon ;  so  that,  if  the  whole 
of  the  45  g.  of  carbon  were  converted  into  sugar,  1 1 2  g.  of  sugar 
could  be  formed  from  the  100  g.  of  proteid  ;  and  if  this  were  all 
excreted,  the  ratio  between  sugar  excreted  and  nitrogen  excreted 

D  112 

=^t  would  be  ,  or  nearly  7.     He  found  the  ratio  in  dogs  after 

excision  of  the  pancreas  to  be  fairly  constant  at  about  2.8.1  A 
constant  value  for  this  ratio  has  been  found  also  by  Lusk  in 
animals  excreting  sugar  under  the  influence  of  phlorrhizine,  in 
dogs  about  3.7,  but  in  other  animals,  rabbits,  goats,  and  cats,  about 
the  same  as  that  found  by  Minkowski  in  his  experiments.  In 
diabetes,  however,  the  ratio  may  have  any  value,  up  to  even  1 1.8.2 
Such  a  high  value  as  this,  if  it  is  right  to  suppose  that  the 
nitrogen  is  excreted  as  rapidly  as  the  sugar — an  assumption  that 
has  been  questioned3 — would  point  to  the  possibility  of  sugar 
being  derived  from  the  products  of  fat  metabolism.  Leaving, 
however,  on  one  side  for  the  present  the  question  whether  the 
synthesised  sugar  may  be  derived  from  substances  formed  from 
fat,  and  assuming  that  it  may  be  derived  from  substances 
formed  from  proteid,  it  is  at  once  obvious  that  what  we  have 

1  Minkowski,  S.A.  31,  85,  1892. 

2  Rumpf,  B.  k.  IV.,  No.  9,  1899.    In  fifteen  days,  1 170  g.  of  sugar  and  98.8  g. 
nitrogen  were  excreted  on  a  diet  strictly  confined  to  fat  and  proteid.    Hartogh 
and  Schumm,  S.A.  45,  31,  observed  in  a  dog  treated  with  large  doses  of 
phlorrhizine,  on  a  diet  rich  in  fat  but  practically  free  from  carbohydrate,  the 

ratio  5_  =  9  maintained  for  four  days,  and  on  one  day  as  high  as  13. 

3  O.  Loewi,  S.A.  47,  68,  1901. 


40  SYNTHESIS  OF  CARBOHYDRATES  [LECT. 

learnt  in  recent  years  about  the  presence  of  sugar  or  simple 
derivatives  of  sugar  in  proteid  molecules,  raises  a  very  important 
question.  When  glycogen  or  glucose  is  formed  from  proteids  in 
the  body,  is  this  a  fundamental  synthesis  of  sugar  from  carbon 
and  hydrogen  in  simpler  combinations  which  are  set  free  when 
proteid  molecules  go  to  pieces,  or  is  it  merely  that  ready-made 
sugar  groups  are  picked  out  of  these  molecules  as  such,  preserved 
from  the  general  breakdown,  and  synthesised  into  glycogen  or 
excreted  as  sugar  ?  If  this  latter  conception  of  the  change  can 
be  admitted,  then  the  problem  presented  is  a  comparatively 
simple  one.  When  once  the  carbohydrate  is  set  free  from  the 
proteid,  the  formation  of  sugar  or  glycogen  from  proteid  involves 
changes  no  more  complicated  than  those  by  which  one  carbo- 
hydrate is  in  the  body  convertible  into  others. 

In  order  to  decide  this,  we  must  know  what  the  amount  of 
carbohydrate  in  proteids  is.  The  isolation  of  carbohydrates  or 
their  derivatives  from  the  debris  of  demolished  proteid  molecules 
is  not  easy,  and  a  quantitative  isolation  is  not  possible.  It  is 
possible  to  estimate  the  carbohydrate  from  the  reducing  power 
of  the  fluid  obtained  on  hydrolysis  of  the  proteid ;  but  we  have 
no  means  of  knowing  that  such  estimations  are  reliable ;  for  all 
that  reduces  need  not  all  be  sugar,  and  some  sugar  may  have 
been  destroyed  during  the  operation  of  hydrolysis.  But, 
estimated  in  this  way,  the  glucosamine  in  certain  typical  glyco- 
proteids,  ovomucoid  and  the  mucin  in  bronchitis  sputa,  amounts 
to  about  35  per  cent.,  in  others  from  25  to  30  per  cent,  of  the 
compound.  These  glyco-proteids,  however,  are  proteids  which 
play  but  a  small  part  in  metabolism.  Egg-albumin  by  this 
method  is  found  to  contain  from  10  to  11  per  cent,  serum- 
globulin  less  than  2  per  cent.,  serum-albumin  less  than  i  per 
cent.  In  some  measure  these  values  may  be  perhaps  checked 
by  deducting  from  the  percentage  elementary  composition  of 
these  proteids  the  carbon  and  nitrogen  of  the  glucosamine 
found  in  each,  and  determining  the  ratio  of  carbon  to  nitrogen  in 
the  remaining  true  proteid  moiety.  Thus,  egg-albumin  has 
52.75  percent.  C.  and  15.45  Per  cent.  N.,  glucosamine  40  per 
cent.  C.  and  7.5  per  cent.  N.  In  100  parts  of  this  albumin,  then, 


IL]  GLYCOPROTEIDS  AND  GLYCOSURIA  41 

the  10  per  cent,  of  glucosamine  would  account  for  4  parts  of 
carbon  and  0.75  parts  of  N.,  and  the  90  parts  of  proteid  freed 
from  glucosamine  would  contain  48.75  parts  of  carbon  to  14.7 

£ 

parts  of  N.,  so  that  the  ratio  ^  =  3.31.     Calculated  in  the  same 

C 

way,  the  proteid  part  of  ovomucoid  gives  the  ratio  —  =  3.5,  and  of 

serum-albumin  and  globulin,  3.25  and  3.32  respectively.  The 
values  given  for  the  carbohydrate  in  these  proteids  seem,  there- 
fore, to  be  relatively  pretty  nearly  correct.  At  any  rate  the  mucin 
group  of  proteids  contain  some  30  per  cent,  more  carbohydrate 
than  the  albumin  and  globulin  of  the  blood.  It  is  said  that  the 
proteids  of  cell  protoplasm  do  not  give  the  a-naphthol  reaction 
at  all.  That  would  imply  that  they  were  still  poorer  in  carbo- 
hydrate than  the  blood  proteids.  But,  on  the  other  hand, 
Neuberg  found  that  the  human  liver  freed  from  glycogen  as 
far  as  it  is  possible  to  free  it  by  boiling  with  water,  gives 
glucosamine,  upon  hydrolysis  with  hydrobromic  acid,  amounting 
to  3.6  per  cent,  of  the  solids,  which  is  about  5  per  cent  of  the 
proteids. 

It  is  not  possible  to  say  how  much  too  low  these  estimates 
are,  if  at  all.  But  it  is  at  any  rate  not  fair  to  be  so  influenced  by 
the  fact  that  some  proteids  yield  35  per  cent,  as  to  argue  that  if 
others  yield  I  or  2  per  cent,  this  is  too  low  a  figure.  It  is  to 
differences  of  this  order  that  the  elementary  composition  of  the 
different  proteids  points.  And  there  is  no  sure  basis  whatever 
for  taking  10  per  cent  as  the  average  amount  of  carbohydrate 
in  proteids  generally  throughout  the  body,  as  Pfliiger  proposed. 
The  proteids  that  give  the  high  figures  are  those  that  are  of 
least  importance  in  general  metabolism,  and  those  that  are  most 
important  and  most  abundant  give  the  low  figures.  Even  if  we 
take  Neuberg's  estimate  of  the  glucosamine  in  the  liver,  which 
amounts  to  5  per  cent,  of  the  proteids  in  the  liver,  as  a  typical 
figure  for  tissue  proteids,  this  amount  of  glucosamine  would 

give  a  ratio  —  =  0.3,  or  only  about  a  tenth  of  the  ratio  which 
Minkowski  found.  So  that  it  is  not  possible  to  account  for  the 


42  SYNTHESIS  OF  CARBOHYDRATES  [LECT. 

sugar  in  his  experiments,  and  others  of  the  same  nature,  by 
supposing  that  it  came  from  the  ready-formed  glucosamine 
groups  of  the  proteids  broken  down. 

Besides,  in  so  far  as  it  is  proved  that  proteids  are  the  source 
from  which  the  sugar  in  severe  diabetes  is  derived,  it  is  proved 
that  the  proteids  which  contain  no  glucosamine  at  all  form 
sugar,  no  less  than  those  that  contain  it.  In  a  case  of  diabetes, 
Falta  found  that  caseine  caused  an  even  more  pronounced 
degree  of  glycosuria  than  egg-albumin  or  serum-globulin  ;  and 
similarly,  Mohr's  experiments  with  nutrose  and  meat  gave  a  more 
marked  effect  than  egg-white.1 

It  is  even  a  question  whether  glucosamine  should  be  con- 
sidered as  the  equivalent  of  sugar  in  the  body  at  all.  No  direct 
evidence  at  any  rate  of  the  formation  of  glycogen  from  gluco- 
samine has  been  obtained.  It  has  been  administered  to  starving 
animals,  as  the  hydrochloride  and  as  the  free  base,  but  in 
neither  case  did  it  appear  to  have  increased  the  amount  of 
glycogen  in  the  liver,  since  animals  to  which  it  was  not  given, 
but  which  were  starved  for  the  same  period,  had  no  less 
glycogen  in  their  livers.2  But  this  method  of  experimenting 
does  not  give  very  sharp  results,  since,  as  Pfliiger  has  pointed 
out,  the  amount  of  glycogen  varies  very  much  in  different 
animals  starved  for  the  same  length  of  time.  And  Lang  has 
shown  that  glucosamine  gives  up  ammonia,  just  as  the  amido 
acids  do,  in  the  presence  of  substances  contained  in  the  cells  of 
many  organs,  including  the  liver  and  intestines.3  If  the  removal 
of  ammonia  by  hydrolysis  is  the  only  change  brought  about  in 
the  glucosamine,  then  certainly  glucose  must  be  the  product  of 
the  change. 

If,  therefore,  we  are  to  look  to  proteids  at  all  as  the  source 
from  which  the  material  for  the  formation  of  sugar  in  the  body 
is  derived,  it  can  hardly  be  to  any  considerable  extent  the  ready- 
made  carbohydrate  groups  in  certain  of  these  proteids  that 
account  for  this  sugar.  The  formation  of  sugar  from  proteids  is 

1  Cf.  Ergeb.  iii.,  p.  493  ;  and,  Mohr,  Z.f.  k.  J/.,  1904. 

2  Cf.  Cathcart,  H.-S.  Z.  39,  423,  1903. 

3  Lang,  H.  B.  v.,  340,  1904. 


ii.]  LEUCINE  AS  A  SOURCE  OF  SUGAR  43 

a  problem  that  cannot  be  so  easily  disposed  of;  a  synthesis  of  a 
really  fundamental  nature  must  be  involved. 

Leucine  was  the  first  of  the  derivatives  of  proteids  to  be 
thought  of  as  a  possible  source  of  sugar.  It  is  the  most 
abundant  of  the  amido  acids  to  be  obtained  from  proteids.  It 
has  a  chain  of  six  carbon  atoms,  though  this  chain  is  not  normal 
but  branched  ;  and  it  is  perhaps  conceivable  that  after  removal 
of  the  nitrogen,  by  oxidation  of  one  of  the  hydrogen  atoms 
attached  to  each  of  the  carbon  atoms,  and  by  reduction  of  the 
carboxyl  to  the  aldehyde  group,  and  lastly,  by  conversion  of  the 
branched  chain  into  a  normal  one,  leucine  might  be  converted 
into  sugar  without  being  first  broken  up.  But  that  any  one  of  this 
series  of  changes  does  take  place,  we  have  no  evidence  whatever. 
The  difficulties  are  not  removed  by  pointing,  as  has  been  done, 
to  the  formation  of  saccharin ic  acid  with  a  branched  chain  from 
glucose  with  a  straight  normal  chain,  which  is  brought  about  by 
the  action  of  lime-water.  Saccharinic  acid, 


C.OH.C/          .     Gf          .     CH2OH 


COOH/  NOH        x)H 

does  not  bring  us  very  near  to  leucine, 


5\CH  .  CH0 .  C/  COOH 

CH/ 


And  the  tetra-oxyamido-caproic  acid  obtained  by  Orgler  and 
Neuberg  from  cartilage,1  which  has  also  been  quoted  in  support 
of  the  conversion  of  leucine  into  sugar,  is  a  substance  the  exact 
constitution  of  which  is  not  known.  If  it  were  known,  it  is 
questionable  whether,  from  what  is  at  present  known  of  its 
occurrence,  it  would  be  reasonable  to  infer  that  it  was  an  inter- 
mediate stage  in  a  reaction  widely  prevalent  in  the  body  by 
which  leucine  could  be  transformed  into  sugar.  Leucine,  in 
fact,  suggests  itself,  before  all  other  proteid  cleavage  products,  as 

1  Orgler  and  Neuberg,  //.-5.  Z.  37,  107,  1903. 


44  SYNTHESIS  OF  CARBOHYDRATES  [LECT. 

a  precursor  of  sugar  only  because  of  its  six  carbon  atoms.  And 
the  "hexone  bases,"  the  bases  containing  six  carbon  atoms, 
lysine,  arginine,  and  histidine,  are  still  less  likely  to  preserve 
their  six  carbon  atoms  in  one  molecule  in  any  changes  leading 
to  the  formation  of  sugar.  Attempts  to  prove  that  leucine  gives 
rise  to  sugar  in  the  body  have  not  been  conclusive.  Rabbits 
have  been  kept  without  food  for  from  four  to  six  days,  and  then 
given  20  g.  or  more  of  leucine  ;  some  hours  later  more  glycogen 
has  been  found  in  the  liver  in  one  or  two  cases  than  in  that  of 
control  animals  that  had  had  no  leucine.1  Rabbits,  fed  with 
leucine  after  severe  strychnine  spasms,  have  been  found  to  have 
no  hepatic  glycogen.2  On  the  other  hand,  Mohr  found,  in  a  case 
of  severe  diabetes  kept  on  a  constant  diet,  that  the  administra- 
tion of  20  g.  of  leucine  increased  the  daily  excretion  of  sugar  to 
the  extent  of  about  10  or  15  g.3 

Of  the  other  cleavage  products  of  proteids,  the  one  which  has 
been  regarded  as  likely  to  be  specially  concerned  in  the  produc- 
tion of  sugar  is  alanine.  By  the  substitution  of  hydroxyl  for  the 
amido  group  in  alanine,  lactic  acid  would  be  formed.  Lactic 
acid  is  known  to  be  very  readily  formed  from  sugar  under 
many  different  conditions.  It  is  possible  that  the  opposite 
change  from  lactic  acid  to  sugar  may  occur.  Neuberg  and 
Langstein  gave  alanine  to  rabbits  that  had  been  kept  without 
food  for  eleven  days,  and  found  lactic  acid  in  the  urine.  In  the 
liver  they  found  glycogen  amounting  to  from  one  to  two 
grammes — more  than  might  be  expected  after  so  long  a  period  of 
starvation,  but  not  more  than  may  be  accounted  for  without 
supposing  a  direct  transformation  of  alanine  into  glucose.4 
Kraus  estimated  by  Pfliiger's  method  the  amount  of  glycogen  in 
five  cats  that  had  been  similarly  fed  for  some  time :  he  found 
the  glycogen  to  be  on  an  average  0.31  per  cent,  of  the  weight  of 
the  cats  ;  the  maximum  being  0.47,  and  the  minimum  0.20  per 
cent.  Five  other  cats  also  similarly  dieted  were  then  treated  with 

1  Cohn,  H.-S.  Z.  28,  210,  1899. 

2  Simon,  ff.-S.  Z.  35,  320,  1902. 

3  Mohr,  Z.f.k.  M.,  1904. 

4  Neuberg  and  Langstein,  D.  /?.  A.,  supp.,  p.  514,  1903. 


i,.]  ALANINE  AND  ASPARTIC  ACID  45 

phlorrhizine,  and  kept  without  food  for  from  five  to  eight  days, 
when  they  were  killed.  The  sugar  that  had  been  excreted  in 
this  time  together  with  the  glycogen  still  remaining  in  the 
bodies  of  the  cats  amounted  to  0.67  per  cent  of  their  weight. 
The  maxima  were  1.23  and  0.77  per  cent,  given  by  two  animals 
to  which  5  g.  of  alanine  had  been  given  daily :  the  minimum 
was  0.33  per  cent,  in  an  animal  that  had  had  I  g.  of  leucine 
daily.1  Embden  and  Salomon  experimented  on  dogs  with 
glycosuria,  following  removal  of  the  pancreas.  In  one  case  34  g. 
of  alanine  were  given,  and  14  g.  of  sugar  above  the  amount 
excreted  with  constant  diet  were  found  in  the  urine  of  the  next 
two  days.  Another  dog  kept  without  food  excreted  daily  for 
four  days  on  an  average  3.5  g.  of  sugar,  and  then  in  the  two 
days  following  a  dose  of  20  g.  of  alanine  excreted  25  g.2  They 
also  found  that  sodium  lactate  increased  very  considerably  the 
output  of  sugar  in  animals  with  pancreatic  diabetes.3  The 
amount  of  alanine  that  can  be  obtained  from  proteids  is, 
it  is  true,  but  small ;  but  it  is  remarkable  that  compounds 
of  alanine  are  numerous ;  tyrosine,  phenyl-alanine,  trypto- 
phane,  cystine,  serine,  histidine,  are  all  compounds  of  alanine, 
and  it  is  possible  that  from  some,  at  any  rate,  of  these  alanine 
may  be  available  for  the  synthesis  of  sugar. 

Another  amido  acid  for  which  there  is  the  same  sort  of 
evidence,  that  it  can  undergo  changes  ultimately  leading  to  the 
formation  of  sugar,  is  aspartic  acid.  Embden  and  Salomon 
found  that  in  their  dogs  a  dose  of  20  g.  of  aspartic  acid  increased 
the  sugar  excretion  by  six  or  seven  grammes.  A  similar  result 
was  observed  by  Knopf  in  phlorrhizine  glycosuria.4  And  twenty 
years  ago  Rohmann  noted  that  rabbits  fed  on  carbohydrates 
and  asparagine  always  had  more  hepatic  glycogen  than  others 
fed  on  carbohydrates  alone,  up  to  even  eleven  times  the  amount.5 
It  would  be  possible  to  connect  the  conversion  of  asparagine  or 

1  F.  Kraus,  D.  M.  IV.  14,  1903  ;  and,  B.  k.  W.  i,  4,  i9°4- 

2  Embden  and  Salomon,  H.  B.  v.,  508,  1904. 

3  H.  B.  vi.,  63,  1904. 

4  Knopf,  S.  A.  49,  135,  1903  ;  cf.,  too,  Nebelthau,  M.  M.  W.  917,  1902. 
6  Rohmann,  Cbl.f.  k.  M.  35,  2,  1884. 


46  SYNTHESIS  OF  CARBOHYDRATES  [LECT. 

aspartic  acid  into  sugar  with  that  of  alanine,  if  it  were  shown 
that  aspartic  acid  give  up  CO2  in  the  body,  thus : 

COOH.CH9.C/       —    COOH ^CO2  +  CH3.C/      —COOH 

NsTH2  \NH2 

Such  a  change  is  not  without  parallel ;  and  if  it  occurs,  then  in 
this  case  too  the  formation  of  sugar  from  proteid  cleavage 
products  may  be  conceived  of  as  occurring  through  lactic  acid  as 
an  intermediate  stage. 

If  these  results  be  taken  to  point  to  lactic  acid  as  a  probable 
stage  in  the  synthesis  of  sugar  in  animals,  then  it  is  important 
in  this  connection  to  take  note  of  the  striking  result  obtained 
by  Luthje  with  glycerine.  Glycerine  is  a  substance  which  has 
certain  relationships  with  lactic  acid  in  biological  chemistry. 
Glyceric  aldehyde, 

/H  ,Q 

Q/          £<y 

>H  "\OH  '\H 

is  isomeric  with  lactic  acid,  and  from  glyceric  aldehyde  it  is 
known  that  sugar  can  be  synthesised.  Luthje  fed  a  dog,  that 
had  had  its  pancreas  removed,  with  large  quantities  of  glycerine 
mixed  with  serum.  In  fourteen  days  the  dog  took  2890  c.c.  of 
glycerine,  and  during  this  period  1408  g.  of  sugar  were  passed 
in  the  urine.  The  dog  weighed  to  begin  with  15.2  kg.,  so  that 
if  there  were  in  its  body  the  maximum  amount  of  glycogen 
ever  found  in  a  dog,  the  equivalent  of  41  g.  of  sugar  per  kg., 
then  623  g.  of  sugar  could  have  been  formed  from  the  glycogen 
of  the  animal's  body.  This  leaves  785  g.  to  be  accounted  for 
from  the  glycerine  or  from  proteids  or  fats.  The  nitrogen 
excreted  in  the  fourteen  days  amounted  to  210  g.  If  we 
suppose  that  for  each  gramme  of  nitrogen  excreted,  3  g.  of  sugar 
were  formed  from  the  proteid  after  the  removal  of  this  amount 

of  nitrogen ;  if  we  take,  that  is,  the  ratio  —  as  =  3,  which  is  a 

high  value  for  this  ratio,  then  630  g.  of  sugar  may  have  come 
from  the  proteid  broken  down.  That  leaves  155  g.  to  be 


ii.]  GLYCERINE:  LACTIC  ACID:  GLYCOCOLL  47 

accounted  for  from  the  glycerine  or  from  fats.  It  is  not  easy 
to  escape  from  the  conviction  that  glycerine  in  this  case  was 
converted  into  sugar.1 

If,  therefore,  glycerine  and  those  amido  acids  which  may 
give  rise  to  lactic  acid  in  the  body  may  serve  for  the  synthesis 
of  sugar,  it  certainly  seems  as  if  the  synthesis  of  sugar  probably 
is  effected  by  condensation  of  chains  each  containing  three  carbon 
atoms,  as  in  the  well-known  synthesis  of  sugar  from  the  oxidation 
products  of  glycerine,  glyceric  aldehyde  and  dioxy-acetone. 

But  Embden  and  Salomon  obtained  as  marked  results  with 
glycocoll  as  those  quoted  above,  which  they  obtained  with  lactic 
acid,  alanine,  and  aspartic  acid.  If  these  results  are  also  to  be 
taken  as  evidence  for  the  direct  synthesis  of  sugar  from  the 
substance  administered,  then  we  have  no  hypothesis  so  near  at 
hand  to  account  for  a  synthesis  such  as  this  implies.  It  is 
difficult  to  imagine  the  formation  of  lactic  acid  or  any  three 
carbon  chain  from  glycocoll.  P.  Mayer  has  injected  glycollic 
aldehyde, 


•  \  \ 

X)H  XH 

into  rabbits,  and  found  about  10  per  cent,  of  the  theoretical 
amount  of  glucose  in  the  urine,  and  this  he  is  inclined  to 
suppose  was  formed  by  condensation  of  the  aldehyde  directly 
to  sugar.2  Even  if  the  sugar  was  formed  from  the  substance 
injected,  it  is  not  proved  that  the  aldehyde  condensed  without 
undergoing  any  other  change.  We  are  equally  at  liberty  to 
suppose  that  a  sugar  synthesis  occurs  in  the  body,  not  only  from 
this  substance,  but  from  glycocoll  and  from  any  fatty  acid, 
whether  derived  from  amido  acids  or  from  fats,  by  condensation 
of  formic  aldehyde  groups.  Till  we  know  how  the  simple  fatty 
acids  such  as  acetic  acid  are  dealt  with  in  the  body,  we  cannot 
say  whether  it  may  not  be  possible  that  formic  aldehyde  groups 
should  be  snatched  out  of  the  burning  of  these  substances  and 

1  Liithje,  D.  A.f.  k.  M.  80,  98,  1904. 

2  P.  Mayer,  H.-S.  Z.  38,  148,  1903. 


48  SYNTHESIS  OF  CARBOHYDRATES  [LF.CT. 

used  for  the  synthesis  of  sugar  by  a  condensation   similar  to 
that  which  Bayer  supposed  to  be  brought  about  in  plants.1 

Strict  proof,  it  must  be  remembered,  has  not  been  obtained 
that  the  sugar  that  appears  in  the  body,  and  cannot  be  derived 
from  the  carbohydrates  of  the  body,  has  necessarily  been 
derived  from  material  supplied  by  proteids.  It  may  be  that 
the  proteids,  or  proteid  cleavage  products,  which  often  appear 
to  give  rise  to  an  increased  formation  of  sugar,  do  so  in  some 
indirect  way.  Pfliiger,  who  has  subjected  all  the  evidence  for 
the  formation  of  sugar  from  proteids  to  what  will  at  times 
appear  an  unnecessarily  stringent  criticism,  interprets  all  the 
cases  in  which  sugar  appears  to  be  derived  from  proteids,  by 
supposing  that  proteids  and  proteid  cleavage  products  stimulate 
the  liver  to  increased  activity  in  converting  fats  into  carbo- 
hydrates. This  latter  change  is  no  less  difficult  to  understand 
than  the  change  from  proteid  cleavage  products,  so  that  our 
difficulties  are  not  removed  by  this  supposition.  Whether  it  be 
fats  or  proteids,  or  both,  that  supply  the  material  for  sugar 
synthesis,  we  are  for  the  present  brought  up  to  a  stop  by  the 
fact  that  we  cannot  follow  the  reactions  by  which  either  fats  or 
proteids  are  oxidised.  We  know  something  about  the  hydrolytic 
processes  in  which  these  substances  are  involved,  but  in  order 
to  be  able  to  advance  further  we  need  to  know  something  of 
the  later  stages  in  their  breakdown.  At  some  point,  clearly, 
some  substance  or  substances  arise  which  by  a  side-reaction 
can  condense  and  give  rise  to  the  synthesis  of  carbohydrate 
groups.  However  tempting  it  may  be  to  speculate  as  to  the 
nature  of  this  substance,  whether  it  be  formic,  glycollic,  or 
glyceric  aldehyde,  or  any  other  possible  derivative  of  lactic  acid, 
the  fact  remains  for  the  present  that  we  do  not  know  what  the 
nature  of  the  substance  is.  If,  as  we  shall  see  there  is  reason 
for  believing  to  be  the  case,  the  amido  acids  lose  their  amido 
groups,  they  become  simple  fatty  acids  or  non-nitrogenous 
derivatives  of  these,  the  oxy-acids,  and,  according  to  one  view 

1  Dakin  has  shown  that  formic  aldehyde  is  produced  when  glycocoll  is 
oxidised  with  hydrogen  peroxide  and  ferrous  sulphate.— Journal  of  Biological 
Chemistry,  i.,  171,  1906. 


ii.]     THE  REAL  STARTING-POINT  OF  THE  SYNTHESIS    49 

at  any  rate  of  the  breakdown  of  the  higher  fatty  acids,  these 
substances  too  will  be  formed  from  fats.  So  that  it  may  well 
be  that  some  one  and  the  same  simple  non-nitrogenous  com- 
pound may  arise  at  some  point  in  the  course  of  the  reactions  by 
which  both  fats  and  proteids  alike  are  broken  down ;  and  that 
this  substance  is  capable,  under  the  conditions  obtaining  in  the 
body,  of  undergoing  a  synthetic  condensation  to  sugar. 


LECTURE  III 

CARBOHYDRATE   CATABOLISM 

THE  final  products  of  the  metabolism  of  non-nitrogenous 
substances  in  the  body  are  carbonic  acid  and  water.  Physiology 
has  been  much  concerned  with  the  mode  of  origin  of  the  principal 
nitrogenous  substances,  that  are  also  final  products  in  metabolism  ; 
but  in  the  matter  of  the  origin  of  carbonic  acid  and  water,  we 
have  still  to  be  content  with  the  simile  of  the  combustion 
furnace :  the  fats  and  carbohydrates  are  completely  burnt  up, 
and  leave  the  body  as  carbonic  acid  and  water.  There  is 
something,  however,  known  about  the  decomposition  of  sugar 
in  vitro,  and  much  of  this  has  almost  certainly  important 
bearings  on  the  physiology  of  carbohydrates  and  the  course  of 
the  reactions  by  which  in  the  body  they  are  finally  converted 
into  these  end-products.  If  the  view  of  biological  chemistry 
which  is  being  generally  adopted  is  correct,  that  the  reactions 
in  living  organisms  are  not  different  in  kind  from  those  that  can 
be  observed  in  the  same  material  where  no  life  is  present,  but 
that  the  differences  depend  upon  the  acceleration  of  certain 
phases  in  these  reactions,  which  is  effected  by  catalytic  agents, 
then  whatever  can  be  made  out  as  to  the  natural  lines  of  cleavage 
in  the  molecules  of  sugars  and  fats  must  be  of  significance  in  the 
physiology  of  these  substances.  Catalysis,  which  is  essentially 
merely  a  change  in  the  velocity  of  reactions,  may,  it  is  true,  by 
hurrying  these  reactions  past  the  points  in  their  course  at  which 
secondary  reactions  are  prone  to  occur,  acquire  a  directive 

50 


LECT.  HI.]        ACTION  OF  ALKALIES  ON  SUGARS  51 

control,  but  though  it  may  thus  alter  the  yield,  it  must  work 
along  the  lines  which  determine  also  the  general  course  of 
change  in  vitro. 

There  are  many  reasons  why  for  us  special  interest  should 
attach  to  the  familiar  reaction  by  which  sugar  is  converted  into 
lactic  acid.  When  sugar  solutions  are  treated  with  alkalies  at  a 
temperature  of  60-70°  they  rapidly  undergo  changes  which  result, 
except  in  the  case  of  cane  sugar,  in  a  discoloration  of  the  fluid, 
familiar  under  the  name  of  Moore's  test.  What  the  yellow  or 
brown  colour  is  due  to  is  not  known,  but  the  most  abundant 
derivative  from  the  decomposed  sugar,  under  certain  conditions 
at  any  rate,  is  lactic  acid.  That  this  acid  is  formed  from  sugar 
by  alkalies,  was  shown  by  Hoppe-Seyler  and  Schiitzenberger,1 
and  in  1881-2  Nencki  made  a  special  study  of  the  change  and 
its  conditions.2  A  solution  containing  10  per  cent,  of  glucose 
and  20  per  cent,  of  caustic  alkali  after  twenty-four  hours  at 
blood  heat  contains  only  traces  of  sugar,  but  lactic  acid  amount- 
ing to  50  per  cent,  of  the  sugar  taken.  The  conversion  takes 
place  in  the  complete  absence  of  air,  and  is  to  be  observed  not 
only  in  the  case  of  glucose,  but  also  with  lactose,  maltose, 
laevulose,  galactose,  the  pentoses,  arabinose,  and  xylose,3  but  not 
cane  sugar — with  those  sugars,  that  is,  that  contain  the  unaltered 
aldehyde  or  ketone  group,  and  for  that  reason  give  Moore's 
reaction.  Ammonia  and  the  alkaline  carbonates  do  not  produce 
the  change,  but  neurine  and  organic  ammonium  bases  on  the 
other  hand  do.  Duclaux  has  since  then  shown  that  even  at  the 
room  temperature,  in  the  presence  of  sunlight,  baryta  water  forms 
lactic  acid  from  sugar  to  the  amount  of  60  per  cent. ;  and  this 
has  been  confirmed  by  Buchner,  who  also  found  that  even  in  the 
dark,  lactic  acid  was  formed  from  a  5  per  cent,  solution  of  glucose 
in  5  per  cent,  caustic  potash,  amounting  in  eleven  months  to 
15  per  cent,  at  the  ordinary  temperature.4 

1  Hoppe-Seyler,  B.  4,  396,  1871. 

2  Nencki,/.  Pr.  Ch.  24,  498,  and  26,  i,  1881-2. 

3  Katsuyama,  B.  35,  669,  1902. 

4  Duclaux,  A.  P.  L  7,  751,  and  10,  168  ;  Buchner  and  Meisenheimer,  B. 
38,  620,  1905  ;  B.  37,  417, 


52  CARBOHYDRATE  CATABOLISM  [LECT. 

Even  more  familiar  than  this  action  of  inorganic  and 
organic  bases,  is  the  resolution  of  sugar  into  lactic  acid  by  the 
action  of  micro-organisms.  Countless  varieties  of  bacilli, 
including  B.  typhosus  and  B.  coli ;  cocci,  vibrios,  including  the 
cholera  vibrio ;  and  sarcinae,  are  known  to  bring  about  this 
change.  The  bacilli  that  cause  milk  to  curdle  are  capable  of 
forming  as  much  as  80  per  cent,  of  lactic  acid  from  glucose,  so 
that  in  this  case,  even  if  none  of  the  sugar  fails  to  yield  one 
molecule  of  the  acid,  60  per  cent,  of  the  sugar  must  undergo 
changes,  summed  up  in  the  equation — 

C6H1206  =   2C3H603 

In  some  of  the  other  fermentations  of  sugar  by  micro- 
organisms lactic  acid  is  believed  to  be  formed  at  one  stage  of 
the  fermentation.  Pasteur  showed  that  a  bacillus  that  formed 
butyric  acid  from  sugar  formed  it  also  if  lactic  acid  were 
substituted  for  the  sugar.  This  has  not  been  found  to  hold  by 
any  means  for  all  the  micro-organisms  that  cause  butyric 
fermentation,  but  it  is  commonly  and  fairly  assumed  that  those 
which  can  convert  calcium  lactate  into  butyrate,  when  they 
convert  sugar  into  butyric  acid,  do  so  by  first  converting  it  into 
lactic  acid. 

Buchner  has  recently  expressed  the  view  that  also  in  the 
alcoholic  fermentation  of  sugar  by  yeast  the  first  stage  is  the 
conversion  of  the  sugar  into  lactic  acid.  His  reasons  for  this 
belief  are  that  lactic  acid  is  frequently  to  be  found  in  the 
expressed  juice  of  yeast  cells,  and  in  that  case  may  disappear 
during  fermentation  ;  or  on  the  other  hand,  it  may  be  absent  in 
the  juice  originally,  and  then  subsequently  appear  during 
fermentation.  This,  he  suggests,  points  to  the  existence  of  two 
reactions  in  alcoholic  fermentation ;  the  first,  the  conversion  of 
sugar  into  lactic  acid,  and  the  second,  the  formation  of  alcohol 
and  carbonic  acid  from  lactic  acid  ;  the  two  reactions  he  ascribes 
to  two  distinct  enzymes,  the  name  "  zymase  "  being  given  to  the 
enzyme  concerned  in  the  first  stage,  and  "  lactacidase "  to  that 
concerned  in  the  second.  Normally,  both  are  present  and 
almost  equally  active  in  the  yeast  cell,  so  that  the  lactic  acid  is 


in.]  LACTIC  ACID  IN  FERMENTATIONS  53 

used  up  at  nearly  the  same  rate  as  that  at  which  it  is  formed.1 
Harden  and  Young  have  shown  that  the  expressed  juice  of 
yeast  when  filtered  through  gelatine  under  pressure,  is  separated 
into  two  parts,  neither  of  which  alone  has  the  power  of  ferment- 
ing sugar,  while  the  mixture  of  the  filtrate  with  the  residue, 
that  cannot  pass  through  the  filter,  has  this  power  like  the  original 
juice.  But  this  separation  is  not  the  separation  of  Buchner's 
two  enzymes.  The  substance  in  the  filtrate  which  is  necessary  in 
the  fermentation  is  not  altered  by  boiling,  and  the  exact  rela- 
tionship of  the  parts  played  by  the  two  constituents  of  the  zymase 
has  not  yet  been  determined  ;  but  provisionally  it  can  be  com- 
pared to  that  found  by  Magnus  between  the  ferment  and  co- 
ferment,  which  together  produce  the  lipolytic  action  of  liver  cells.2 

Some  support  for  Buchner's  conception  of  the  process  in 
alcoholic  fermentation  may  be  derived  from  the  fact  that  Maze" 
has  shown  that  Eurotiopsis  Gayoni  produces  alcohol  from  lactic 
acid ;  and  perhaps,  too,  from  the  fact  that  Thomas  finds  that 
formic  acid  is  produced  by  yeast,  especially  when  certain 
substances  such  as  urea  or  certain  salts  of  ammonia  are  added 
to  the  media  on  which  it  is  grown.3 

Whatever  the  result  of  the  further  investigations  on  this 
point  may  be,  there  is  no  doubt  that  the  formation  of  lactic  acid 
from  sugar  would  make  the  chemistry  of  both  alcoholic  and 
butyric  fermentation  more  intelligible.  For  lactic  acid  is  an 
a-oxy-fatty  acid,  and  it  appears  to  be  a  general  reaction  for  such 
compounds  to  split  into  formic  acid  and  the  aldehyde  of  the  next 
lower  member  of  the  series  of  acids.  Le  Sueur  has  shown  this 
to  hold  for  all  the  fatty  acids  from  stearic  to  lauric.4  And  in  the 
case  of  lactic  acid,  it  is  well  known  that  in  a  number  of  different 
conditions  the  tendency  to  break  up  in  this  way  is  manifested. 

/H  /H 

CHS.C<  —       COOH >CH3.C<        +   H.COOH 

\OH  ^O 

1  Buchner  and  Meisenheimer,  B.  37,  417  ;  and  B.  38,  620,  1904-5. 

2  Harden  and  Young,  //.  Phys.  32,  i.  ;  Magnus,  H.-S.  Z.  42,  149,  1904. 

3  Maze,  A,  P.  I.  16,  446,  1902  ;  P.  Thomas,  C.  R.  136,  1015,  1903. 

4  Le  Sueur,/  C.  5.,  Dec.  1905. 


54  CARBOHYDRATE  CATABOLISM  [LECT. 

When  heated  to  a  high  temperature,  about  440°  C,  the  change 
occurs  spontaneously  ;  in  the  presence  of  dilute  sulphuric  acid, 
it  occurs  at  I3O°C.  Electrolysis  of  lactic  acid  gives  the  same 
products.  And  the  same  reaction  clearly  underlies  the  fact 
observed  by  Duclaux,  that  whereas  the  weaker  bases,  baryta  or 
lime-water,  acting  on  sugar  in  sunlight,  form  lactic  acid,  the 
stronger  base,  potash,  under  the  same  conditions  forms  alcohol 
and  carbonic  acid.  For  the  formic  acid,  on  breaking  up  into 
hydrogen  and  carbonic  acid,  would  furnish  the  hydrogen  for 
the  reduction  of  the  aldehyde  to  alcohol.1  If  lactic  acid  be 
treated  with  sulphuric  acid  and  manganese  dioxide  or  peroxide 
of  lead,  aldehyde  and  carbonic  acid  are  liberated  ;  the  two 
atoms  of  hydrogen  that  would  otherwise  have  been  set  free 
from  the  formic  acid  being  oxidised  to  water  as  soon  as 
formed. 

If,  therefore,  lactic  acid  is  a  precursor  of  alcohol  and  carbonic 
acid  in  alcoholic  fermentation,  the  changes  undergone  by  sugar 
in  this  process  are  far  more  intelligible  than  when  crudely 
summed  up  in  the  equation  : 

C6H12O6  =   2C2H6O  +  2CO2 

And  similarly  the  equation,  by  which  the  final  results  of  butyric 
fermentation  is  represented, 

C6H1206  =   C4H8O2  +  2H2  +  2C02 

also  becomes  intelligible  if  we  suppose  that  two  molecules  of 
lactic  acid  are  first  formed  from  the  sugar,  and  that  these  break 
up  into  two  molecules  of  acetic  aldehyde  and  two  of  formic 
acid.  For  one  of  the  most  familiar  of  the  changes  to  which 
aldehyde  is  liable  is  the  condensation  of  two  molecules  to  form 
aldol— 


/  .o 

=   CH3C<         —     C 


1  Duclaux,  A.  P.  /.  7,  751,  and  10,  168. 


III.] 


LACTIC  ACID >.  ACETIC  ALDEHYDE 


55 


And    this  aldol,   or   /3-oxybutyric  aldehyde,   by   reacting   with 
two  molecules  of  water,  thus — 


CH9. 


H 
OH 

OH 
,0 

:H,C 

( 

H 

*T 

\H 

PkTJ 

would  give 


CH3 .  CH2 .  CH2 .  COOH  +  2  H2O, 


or  butyric  acid.  Butyric  acid  fermentation,  therefore,  like  alcoholic 
fermentation  of  sugar,  may  reasonably  be  expected  to  depend 
on  the  tendency  of  sugar  to  split  up  into  lactic  acid  ;  so,  too,  with 
acetic  acid  fermentation,  in  which  case  it  may  be  noted  that  the 
acetic  acid  produced  always  contains  some  acetic  aldehyde. 

How  the  formation  of  lactic  acid  from  sugar  is  to  be  ex- 
plained is  not  yet  quite  clear.  From  the  fact  that  galactose 
ferments  less  readily  with  yeast  than  glucose,  fructose,  or  mannose, 
in  all  three  of  which  the  atoms  round  the  two  middle  carbon  atoms 
are  disposed  thus — 

H          OH 
_C    —    C    — 

OH          H 

while  in  galactose  the  arrangement  about  these  two  carbon 
atoms  is 

H  H 

— C    —    C    — 

OH         OH 

it  is  possible  that  the  former  arrangement  is  particularly  favour- 
able to  a  reaction  with  water,  thus — 


HO 
H 


H 
,OH 


—  C< 


56  CARBOHYDRATE  CATABOLISM  [LECT. 

resulting    in    the    formation     of    two    molecules    of   glyceric 
aldehyde, 

H 
CH9OH.C 


or  its  hydrate.  Glyceric  aldehyde  has  been  shown  by  Nef  to 
be  converted  into  lactic  acid,  under  conditions  in  which  glucose 
and  fructose  also  yield  lactic  acid,  with  caustic  soda  at  a  slightly 
raised  temperature.  And  the  formation  of  glyceric  aldehyde 
from  glucose  would  be,  if  reversed,  the  familiar  reaction  by 
which  a-acrose  (/-fructose)  is  formed  from  the  oxidation  products 
of  glycerine : 

/U  /H 

CH2OH.C  .     K       +   CH9OH .  CO .  CH9OH 


/H  /H  /OH 

=    CH9OH .  C<          .     C<          —  C<          —  CO  .  CH2OH 
X>H  X)H          \H 


If  this  is  the  way  in  which  the  chain  of  six  carbon  atoms 
splits  up  into  two  chains  of  three,  the  transformation  of  glyceric 
aldehyde  to  lactic  acid  has  still  to  be  accounted  for.  The 
change  is  precisely  the  same  as  that  from  aldol  to  butyric 
acid  referred  to  above,  and  might  be  explained  in  the  same 
way. 

Nef  has,  however,  proved  that  in  the  conversion  of 
sugar  into  lactic  acid  by  the  action  of  caustic  alkali,  pyruvic 
aldehyde, 

^O 
\H 

is  an  intermediate  product  The  probability  of  the  occurrence 
of  pyruvic  aldehyde  in  the  synthesis  of  imidazol  compounds  in 
the  body  will  be  referred  to  in  a  subsequent  lecture.  Buchner 


HI.]  STAGES  IN  LACTIC  ACID  FORMATION  57 

has  adopted  Nef  s  view x  that  this  substance  represents  a  stage 
in  the  formation  of  lactic  acid,  which  is  thus  regarded  as  occur- 
ring by  the  following  steps  : 

M  ,£> 

(i)     CH2OH.C<  .     Cf  Glyceric  aldehyde. 

X)H         \H 


(2)     CHS .  CO  .  Cf  Pyruvic  aldehyde. 

,OH 

^H  '\OH 


(3)    CH3 .  C<          •     K  Lactic  acid- 


Before  we  leave  the  subject  of  the  significance  of  lactic  acid 
in  the  fermentation  of  sugar,  note  should  be  taken  of  the  fact 
that  in  the  case  of  the  Bacillus  acidi  lactici  the  cell-juice  ex- 
pressed from  the  crushed  bacilli  contains  a  substance  which 
produces  the  same  change  in  sugar  solutions  as  the  bacilli 
themselves.  In  other  words,  the  action  of  the  bacilli  is  proved 
to  be  due  to  an  enzyme  which,  like  the  zymase  of  yeast,  is 
capable  of  acting  when  set  free  from  the  living  cell.2 

Is  there  now  any  evidence  that  such  an  enzyme  is  widely 
distributed,  and  actually  plays  a  part  in  the  carbohydrate 
metabolism  of  animals? 

Lactic  acid  is  found  in  the  muscles,  liver,  spleen,  thymus, 
and  thyroid,  in  the  blood,  and  the  bile,  even  in  urine  under 
certain  conditions  ;  in  disease  of  the  liver,  for  instance,  or  after 
severe  muscular  fatigue.  In  all  cases  this  is  the  optically  active, 
dextro-rotatory  acid.  In  the  intestinal  canal,  fermentation  of 
the  sugar  taken  in  food  is  brought  about  by  bacteria,  resulting  in 
the  formation  of  the  usual  mixture  of  both  stereoisomeric 
modifications. 

That  the  dextro-rotatory  acid  found  in  the  organs  is  also 
derived  from  carbohydrates,  has  been  assumed  by  probably 

1  Nef,  Ann.  335,  pp.  254  and  279  ;  Buchner  and  Meisenheimer,  B.  38, 
620,  1905. 

2  Buchner  and  Meisenheimer,  B.  36,  634,  1903;  Herzog,  H.-S.  Z.  37, 
381,  1903. 


58  CARBOHYDRATE  CATABOLISM  [LECT. 

the  majority  of  physiologists.  The  assumption,  in  the  light  of 
many  of  the  facts  we  have  been  reviewing,  was  almost  inevitable  ; 
but  it  is  an  assumption  that  has  not  lent  itself  readily  to 
positive  proof  as  yet.  The  most  interesting  study  of  the 
formation  of  lactic  acid  in  the  tissues  is  that  of  Magnus  Levy, 
on  the  acids  set  free  during  autolysis  of  the  liver.1  If  the  liver 
is  excised  without  bacterial  infection  and  kept  in  sterilised 
vessels  at  the  body  temperature,  it  acquires  after  a  few  hours 
an  acid  reaction.  This  is  due  to  the  formation  partly  of  the 
volatile  fatty  acids,  acetic  and  butyric,  and  partly  of  lactic  acid. 
By  the  end  of  twenty-four  hours  the  amount  of  these  acids 
formed  by  100  g.  of  liver  may  be  sufficient  to  neutralise  close 
upon  20  c.c.  of  normal  alkali ;  expressed  as  lactic  acid,  that 
means  1.8  g. ;  and  in  fact,  in  one  case  1.5  g.  of  lactic  acid  alone 
was  obtained,  from  this  amount  of  liver  in  twenty-four  hours. 
If,  instead  of  altogether  excluding  micro-organisms,  their  growth 
was  merely  arrested  by  means  of  antiseptics,  chloroform  and 
toluene,  it  is  interesting  to  note  that  weeks  or  months  passed 
before  this  amount  of  acid  could  be  obtained.  As  is  often  the 
case  in  such  experiments,  the  antiseptics  evidently  retard  the 
reaction  under  investigation  very  considerably.  And  perhaps 
one  may  say  that  the  closer  the  relation  borne  by  the  reaction 
to  the  essential  phenomena  of  life,  the  more  likely  is  this  to  be 
the  case.  Antiseptics  are  antiseptic  because  they  interfere 
with  reactions  that  are  indispensable  for  the  maintenance  of 
life. 

Simultaneously  with  the  appearance  of  these  acids,  it  was 
found  that  a  diminution  of  the  glycogen  and  sugar  of  the  liver 
took  place  under  these  conditions.  And  in  five  out  of  eight 
experiments  in  which  Magnus  Levy  made  the  necessary 
analyses,  the  amount  of  carbohydrate  that  disappeared  covered 
and  closely  corresponded  to  the  amount  of  acids  formed.  In 
the  three  others,  however,  the  acids  were  in  excess.  This  may 
mean  that  some  of  the  acids,  perhaps  only  one  of  them,  was 
derived  from  some  other  source ;  the  acetic  acid,  for  instance, 
conceivably  from  glycocoll,  or  the  lactic  acid  from  other  proteid 
1  Magnus  Levy,  H.  B.  2,  261,  1902. 


in.]  ORIGIN  OF  LACTIC  ACID  IN  ANIMALS  59 

cleavage  products,  alanine  or  its  compounds  ;  or  possibly  that 
carbohydrate  was  formed  during  the  incubation ;  and  from  this, 
as  from  that  which  was  detected  in  the  control  analyses,  acids 
were  derived. 

It  is  evident  that  these  experiments,  interesting  as  they  are, 
do  not  prove  that  the  lactic  acid  which  is  formed  is  formed 
from  the  sugar  which  disappears.  The  fact,  however,  that 
hydrogen  and  carbonic  acid  are  simultaneously  given  off  by 
the  liver  under  these  conditions  is,  at  any  rate,  in  agreement 
with  the  conception  that  the  butyric  acid  is  derived  from  the 
carbohydrate ;  or,  according  to  the  view  referred  to  above,  con- 
cerning the  origin  of  butyric  acid  in  fermentation,  from  part 
of  the  lactic  acid.  But  it  is  a  common  belief  that  the  lactic 
acid  which  is  found  in  various  organs,  in  the  blood,  and  in 
abnormal  conditions  in  the  urine,  is  not  derived  from  carbo- 
hydrate, but  from  proteid.  This  idea  was  first  indicated  by 
some  observations  on  the  lactic  acid  formed  in  muscles  during 
rigor  mortis.  Boehm  in  1880  found  that  the  lactic  acid  in 
muscles  increased  considerably  during  the  first  hours  after 
death,  but  that  the  glycogen  was  unaffected  so  long  as  bacterial 
decomposition  was  prevented.1  If  that  is  so,  it  is  clear  that  the 
lactic  acid  is  not  derived  from  glycogen.  This  result  has  been 
confirmed  by  Monari,2  and  upheld  by  Boehm  against  the  contrary 
result  obtained  in  Heidenhain's  laboratory  by  Werther.  The 
latter  found  in  aseptic  experiments  that  the  glycogen  in  the 
muscles  diminished  from  0.24  to  0.003  Per  cent-  in  three  hours. 
Kiilz  also  maintains  that  in  the  first  three  or  four  hours 
after  death  the  glycogen  in  the  muscles,  if  the  temperature  is 
not  allowed  to  fall,  is  very  considerably  diminished.3  How  this 
discrepancy  is  to  be  explained  is  not  clear.  Morishima  in 
Boehm's  laboratory  studied  the  action  of  arsenious  acid  on 
cats,  and  showed  that  the  amount  of  lactic  acid  in  the  liver, 
blood,  kidney,  and  intestines  was  increased  sometimes  50, 
sometimes  250  per  cent.  The  glycogen  disappeared  at  the  same 

1  Boehm,  Pfl.  A.  33,  44,  1880. 

2  Monari,  M. .  J.  303,  1889. 

3  Werther,  Pfl.  A.  46,  63,  1889. 


60  CARBOHYDRATE  CATABOLISM  [I.ECT. 

time  almost  entirely.  But  he  argues  for  the  view  that  the 
lactic  acid  is  derived  from  the  proteid,  and  not  from  the 
glycogen  that  disappears,  partly  because  there  is  not  sufficient 
lactic  acid  to  account  for  the  amount  of  glycogen  that 
disappears.1 

The  main  foundation  for  the  belief  that  lactic  acid  is  derived 
from  proteid  in  the  body,  is  Minkowski's  experiments  on  the 
removal  of  the  liver  in  geese.  In  this  condition  lactic  acid 
and  ammonia  very  largely  replace  uric  acid  in  the  urine.  And 
the  amount  of  lactic  acid  is  increased  by  a  proteid  diet,  and 
not  by  carbohydrates.  It  has  been  justly  pointed  out  that  this 
does  not  prove  that  the  lactic  acid  is  a  product  of  proteid 
metabolism,  produced,  as  the  figures  show,  in  equimolecular 
quantities  with  the  ammonia.  The  removal  of  the  liver 
prevents  the  ammonia  from  being  converted  into  neutral  urea, 
and  consequently  this  ammonia  combines  with  lactic  acid,  which 
is  apparently  available  in  considerable  quantities,  and  the  amount 
of  lactic  acid  excreted  is  determined  by  the  amount  of  ammonia 
that  has  to  be  neutralised.2  Besides,  if  the  lactic  acid  excreted 
were  merely  that  which  the  liver  failed  to  convert  into  uric  acid, 
then  there  should  be  no  more  than  one  molecule  of  lactic  acid 
for  four  of  ammonia. 

Other  experimental  results  which  have  been  interpreted  in 
favour  of  the  formation  of  this  acid  from  proteids  are  those  of 
Asher  and  Jackson.  After  removing  the  abdominal  viscera  in 
dogs  they  found  that  lactic  acid  accumulated  in  the  blood,  and 
at  the  same  time  the  nitrogenous  substances  not  precipitated 
from  the  blood  by  alcohol  increased.  These  nitrogenous  sub- 
stances are  taken  as  a  measure  of  proteid  breakdown.3  But  if 
the  amount  of  nitrogen  of  this  form  added  to  100  c.c.  of  blood 
during  the  several  experiments  is  compared  with  the  amount 
of  lactic  acid  found  to  be  formed  at  the  same  time,  it  is  clear 
that  the  relation  is  one  that  is  hardly  compatible  with  the 

1  Morishima,  S.  A.  43,  217,  1900. 

2  Minkowski,  S.  A.  21,  69,  1886;  cf.  Magnus   Levy,   H.  B.  ii.,  p.  283, 
1902. 

3  Asher  and  Jackson,  Z.f.  B.  41,  393,  1901, 


III. 


LACTIC  ACID  FROM  SUGAR  OR  PROTEID? 


61 


formation  of  all  the  lactic  acid  from  the  proteid  broken  down. 
The  figures  are  : — 


Experiment. 

Proteid  broken  down 
=  mg.  N.  found  x  0'25. 

Lactic  Acid  in  mg. 

IV. 

106 

72 

V. 

82 

96 

VI. 

57 

40 

VII. 

94 

43 

VIII. 

66 

64 

IX. 

55 

106 

Mean 

Proteid     77  =  12.3  N. 

Lactic  acid  70  =  90  p.  c.  of 
Proteid  broken  down. 

Putting  this  in  the  form  of  the  ratio  of  lactic  acid  to  the  nitrogen 
set  free  from  proteid,  the  value  obtained  is  5.8,  or  about  three- 
quarters  of  the  theoretical  yield  if  all  the  carbon  of  the  proteid 
were  converted  into  lactic  acid,  which  is  very  hard  to  suppose 
possible. 

None  of  the  arguments  for  the  derivation  of  the  lactic  acid 
in  the  body  from  proteids,  to  the  exclusion  of  carbohydrates, 
are  therefore  satisfactory ;  but  if,  with  Hoppe-Seyler  and 
Nencki,  we  prefer  to  trace  the  lactic  acid  to  a  carbohydrate 
source,  this  need  not  mean  that  we  refuse  to  admit  the  possibility 
of  small  quantities  of  lactic  acid  coming  from  proteid.  It  is 
extremely  probable  that  some  lactic  acid  at  any  rate  is  formed 
in  the  course  of  proteid  metabolism — from  the  alanine,  if  from 
no  other  groups  in  the  proteid  molecules. 

It  is  perhaps  in  the  metabolism  of  muscle  that  lactic  acid 
has  been  thought  to  be  of  most  significance,  and  that  a  relation- 
ship between  it  and  the  carbohydrates  of  the  tissue  has  been 
most  frequently  traced.  There  is  no  doubt  that  some  acid 
substance  or  substances  are  formed  during  the  activity  of 
muscle,  and  there  is  no  doubt  that  the  glycogen  diminishes  at 


62  CARBOHYDRATE  CATABOLISM  [LECT. 

the  same  time  at  a  rate  that  varies  with  this  activity,  till  in 
the  spasms  of  strychnine  poisoning  it  entirely  disappears.  So 
far,  there  has  been  indeed  a  singular  unanimity.  But  the 
question  whether  the  formation  of  lactic  acid  accounts  for 
both  the  acid  reaction  and  the  disappearance  of  the  carbo- 
hydrate, as  there  has  been  a  tendency  to  assume,  cannot  be 
said  to  be  clearly  decided.  The  experiments  on  this  point 
have  for  the  most  part  been  carried  out  in  two  ways.  Either 
the  amount  of  lactic  acid  left  in  the  muscles  after  exhausting 
activity  has  been  estimated,  and  compared  with  the  amount 
found  in  muscles  kept  at  rest,  or  changes  have  been  looked 
for  in  the  amount  of  lactic  acid  present  in  the  blood  coming 
from  muscular  parts.  In  the  latter  case  the  results  have  been 
fairly  congruous,  v.  Frey  and  Gruber  perfused  blood  through 
the  hind-limbs  of  dogs,  and  tetanised  the  muscles.  The  blood 
was  found  to  contain  more  lactic  acid  ;  in  one  case,  after  three 
hours,  as  much  as  ten  times  the  amount  originally  present.1 
Berlinerblau  in  Nencki's  laboratory  obtained  similar  results  in 
similar  experiments  ;  but  also  observed  that  if  sugar  or  glycogen 
was  added  to  the  blood,  the  amount  of  lactic  acid  was  greater 
than  otherwise.-  In  Hoppe-Seyler's  laboratory  Spiro  had 
previously  shown  that  the  blood  of  tetanised  animals  contained 
considerable  quantities  of  lactic  acid,  and  also  that  human  urine 
after  muscular  exertions  contains  lactic  acid.  This  latter  point 
has  been  confirmed  by  others  since,  and  also  shown  to  hold 
for  the  urine  of  frogs.3  Hoppe-Seyler's  opinion  was  that  the 
lactic  acid  was  found  in  the  blood  or  urine  only  when  the 
exertion  had  been  so  severe  that  complete  oxidation  had  been 
interfered  with ;  the  sugar  or  glycogen,  from  which  the  lactic 
acid  was  formed,  would  under  ordinary  conditions  have  been 
completely  oxidised.  Zillessen,  one  of  his  pupils,  found  that 
if  the  arteries  to  the  hind-legs  of  dogs  were  ligatured  for  some 
hours,  and  then  the  blood  allowed  to  flow  into  them  again,  the 

1  v.  Frey,  D.  R.  A.  533,  1883. 

2  Berlinerblau,  S.  A.  23,  333,  1887. 

3  Spiro,  H.-S.  Z.  i,  in,  1877  ;  Colasanti  and  Moscatelli,  M.  /.,  p.  212, 
1887  ;  Werther,  Pfl.  A.  46,  1890. 


in.]  LACTIC  ACID  IN  THE  MUSCLES  63 

blood  collected   from   the  corresponding  veins  contained  more 
lactic  acid  than  that  from  other  parts  of  the  body.1 

In  this  group  of  experiments,  then,  the  evidence  is  clearly 
in  favour  of  the  formation  of  lactic  acid  in  muscular  activity. 
The  other  method  of  investigating  this  question  has  given 
discordant  results.  Berzelius  found  that  the  muscles  of  hunted 
animals  contained  large  amounts,  while  those  of  paralysed  limbs 
contained  very  little  lactic  acid.  In  Heidenhain's  laboratory 
Marcuse  tetanised  one  leg  of  a  number  of  frogs,  and  left  the 
other  leg  at  rest.  In  every  case  the  tetanised  limbs  contained 
less  glycogen  and  more  lactic  acid  than  those  that  had  not 
been  tetanised.2  But  the  results  obtained  by  others  have  been 
the  exact  opposite  of  these,  so  far  as  the  lactic  acid  is 
concerned.3 

Before  we  attempt  to  interpret  these  results,  it  should  be 
made  quite  clear  that  the  reaction  we  are  considering,  by  which 
sugar  becomes  lactic  acid,  is  not  one  of  those  in  which  energy 
is  set  free,  and  therefore  not  one  of  those  metabolic  changes 
by  which  the  muscles  are  able  to  do  work  or  produce  heat. 
The  heat  of  combustion  of  lactic  acid  has  not  been  directly 
determined  ;  that  of  ethyl  lactate  has,  however,  and  for  I  g.  mol. 
is  656  cal.  Deducting  from  this  the  equivalent  of  i  mol.  of 
ethyl  alcohol,  viz.,  325  cal.,  that  gives  us  the  approximate  value 
for  the  equivalent  of  I  g.  mol.  of  lactic  acid  331,  or  of  2  g.  mols. 
662  cal. :  the  equivalent  of  I  g.  mol.  of  glucose  is  677  cal.  In 
another  way  we  may  come  to  a  similar  result.  If  we  suppose 
that  lactic  acid  is  formed  from  sugar,  and  that  it  subsequently 
breaks  down  into  acetic  aldehyde  and  formic  acid,  even  this 
further  hypothetical  reaction  leaves  the  energy  much  as  it  was. 
For  i  g.  mol.  of  glucose  would  on  this  supposition  give  2  g. 
mols.  of  formic  acid  (2  x  61.7  =  123  cal.)  and  2  g.  mols.  of 
aldehyde  (2  x  275.5  =  551  cal);  and  the  sum  of  the  heat 
equivalents  of  these  products  is  674  cal. ;  so  that  even  if  the 


1  Zillessen,  ff.-S.  Z.  15,  392,  1891. 

2  Marcuse,  Pfl.  A.  39,  425,  1886. 


3  Astaschevsky,  H.-S.  Z.  4,  397,  1880;  Warren,  Pfl.  A,  24,  391,  1881  ; 
and,  Monari,  M.  /.  303,  1889. 


64  CARBOHYDRATE  CATABOLISM  [LECT. 

reaction  were  carried  one  step  further  than  lactic  acid,  there 
would  still  be  no  evolution  of  energy.  The  physiological 
significance  of  the  appearance  of  lactic  acid  in  muscle  is  not 
that  which  is  sometimes  assigned  to  it. 

If,  therefore,  the  formation  of  lactic  acid  from  sugar  is  a 
constant  normal  factor  in  the  metabolism  of  muscle,  it  is  merely 
preliminary  to  subsequent  oxidation  processes  in  which  the 
energy  of  the  sugar  serves  for  purposes  of  work  or  heat  pro- 
duction in  this  tissue.  We  should  not  look  on  lactic  acid  as 
a  waste  product  excreted  by  the  muscle  as  useless  and  done 
with,  or  even  as  necessarily  the  product  of  a  miscarriage  of 
metabolism,  as  Hoppe-Seyler  seemed  to  imply.  It  may  be 
that  the  appearance  of  lactic  acid  in  the  blood  and  urine  after 
exertion  is  due  to  the  escape  of  but  a  very  small  part  of  the 
whole  amount  of  this  substance  that  has  been  produced  in  the 
course  of  the  ordinary  metabolism  of  the  muscles,  and  that 
it  has  escaped  merely  because  the  other  subsequent  stages  of 
sugar  catabolism  have  lagged  behind  this  earlier  one ;  a  sign 
perhaps  of  overwork,  or  of  inadequate  oxygen  supply,  as  Hoppe- 
Seyler  himself  supposed.  Such  a  conception  of  the  position 
of  lactic  acid  in  the  chemistry  of  muscle  may  help  to  make 
intelligible  the  discrepant  results,  too,  which  have  been  obtained 
with  excised  muscles,  in  which  excitation  has  sometimes  caused 
a  diminution,  sometimes  an  increase  of  lactic  acid  in  the  tissue. 
Such  results  are  exactly  comparable  with  the  observations  of 
Buchner  on  the  production  of  lactic  acid  in  yeast  juice :  in 
some  samples  he  found  the  lactic  acid  would  increase,  in  others 
it  would  diminish.  If  lactic  acid  is  not  a  final  product  of  the 
metabolism  of  muscle  tissue — neither  the  raw  material  nor  the 
finished  article — it  ceases  to  be  strange  that  the  amount  of 
material  in  a  half-worked  condition  found  in  stock  at  any 
moment  should  differ  in  different  cases.  In  the  series  of 
reactions  by  which  sugar  is  broken  down,  if  the  first  phases  to 
fail  are  those  that  come  earliest,  then  there  must  be  a  diminu- 
tion of  lactic  acid  ;  if,  on  the  other  hand,  the  later  stages  come 
to  a  stop,  while  the  earlier  ones  are  still  actively  carried  on, 
then  there  will  be  an  increase. 


in.]  GLYCOLYSIS  IN  THE  BLOOD  65 

There  is,  therefore,  reason  for  connecting  the  lactic  acid 
found  in  the  organs  we  have  been  considering  with  the  sugar 
or  glycogen  observed  to  disappear  from  them.  The  disappear- 
ance of  sugar  from  body  fluids  or  tissues  has  been  the  subject 
of  a  number  of  important  studies,  in  which  even  the  temporary 
appearance  of  lactic  acid  in  the  place  of  the  sugar  has  not  been 
definitely  determined.  The  observations  from  which  all  these 
investigations  upon  the  phenomena  of  glycolysis  started  were 
made  by  Claude  Bernard.  He  found  that  the  sugar  in  the 
blood  diminished  very  rapidly  in  the  first  hours  after  the  blood 
was  withdrawn  from  the  vessels.  In  the  first  five  hours  60  per 
cent,  of  the  original  amount  of  sugar  might  disappear,  and  at 
the  end  of  twenty-four  hours  none  might  be  left.  Pavy  confirmed 
Bernard's  results,  and  Lepine  more  recently  developed  out  of 
them  and  his  own  experiments  on  this  point  a  theory  of  the 
disorder  of  metabolism  in  diabetes.1  He  found  that  the  glyco- 
lytic  properties  of  the  blood  resided  in  the  leucocytes,  since 
the  blood  corpuscles  were  more  active  than  the  serum,  and  the 
lymph  than  the  blood.  But  the  glycolytic  agent  could  be 
extracted  with  normal  saline,  so  he  regarded  it  as  an  enzyme. 
This  enzyme  he  thought  at  first  was  formed  in  the  pancreas, 
for  three  reasons.  First  of  all,  the  glycolytic  power  of  the 
blood  was  diminished  by  removal  of  the  pancreas.  (This  has 
not  been  found  to  be  the  case  by  others  who  have  repeated 
the  experiments.)  Then  it  was  increased  by  tying  the  duct 
of  the  pancreas  or  cutting  its  nerves.  And  thirdly,  the  blood 
of  the  pancreatic  veins  was  more  active  than  that  of  the  splenic 
vein.  (But  this  also  has  been  disputed  by  others.)  These  ex- 
periments, and  the  attempt  to  derive  from  them  a  complete 
theory  of  the  causation  of  diabetic  glycosuria,  have  led  to  a 
large  number  of  investigations  upon  the  glycolytic  action  of 
the  blood  and  tissues,  and  the  relations  of  the  pancreas  to  this 
action. 

Claude  Bernard  himself  thought  that  the  sugar  was  con- 
verted into  lactic  acid.  Seegen  could  not,  however,  detect  any 
increase  in  the  amount  of  this  substance  present  in  shed  blood. 

1  Lepine,  M.J.,  1891  to  1896. 

E 


66  CARBOHYDRATE  CATABOLISM  [LECT. 

So  that  if  lactic  acid  was  formed  it  must  be  formed  only  as 
an  intermediate  product,  and  must  itself  undergo  further  change. 
If  this  further  change  should  be  of  the  same  nature  as  that 
which  Buchner  believes  to  be  set  up  in  the  lactic  acid  produced 
by  yeast,  then  it  should  be  possible  to  demonstrate  the  forma- 
tion of  carbonic  acid  and  also  of  alcohol.  Oppenheimer  accord- 
ingly tried  to  determine  whether  alcohol  was  formed,  but  could 
get  no  evidence  of  it  except  traces  of  some  substance  that  gave 
the  iodoform  reaction,  but  was  not  acetone.  At  the  same  time, 
he  failed  to  detect  any  evidence  of  the  formation  of  lactic  acid. 
Very  similar  results  were  obtained  by  Herzog,  not  only  with 
blood  but  also  with  the  pancreas.  Blumenthal  found  that  the 
juice  expressed  by  hydraulic  pressure  from  a  variety  of  organs 
developed  carbonic  acid  when  mixed  with  sugar  solutions ;  but 
he  could  not  find  any  alcohol,  till  Feinschmidt  in  his  laboratory 
subsequently  isolated  a  small  quantity  of  alcohol  produced  by 
the  action  of  the  fluid  expressed  from  the  liver  on  sugar  solu- 
tions in  the  presence  of  antiseptics.  In  some  cases  a  precipitate 
obtained  from  the  expressed  juice  by  means  of  alcohol  and 
ether  was  dried,  and  the  solutions  of  this  precipitate  were  found 
to  give  somewhat  better  results  than  the  juice  itself.  In  the 
meantime,  Stoklasa  and  others  working  with  him  found  that 
various  organs,  especially  the  pancreas,  but  also  the  liver  and 
muscles,  contained  a  substance,  precipitated  with  alcohol  and 
ether  from  the  expressed  cell  juice,  which  produced  in  an 
atmosphere  of  hydrogen  a  vigorous  fermentation  when  added 
to  sugar  solutions.  Feinschmidt  in  part  confirmed  Stoklasa's 
results,  but  others  have  either  completely  failed  to  do  so,  or 
shown  that  the  fermentation  which  they  obtained  was  due  to 
bacteria.1 

The  glycolytic  action  of  the  fluid  expressed  from  muscles 
is  said  by  Cohnheim  to  be  elicited  only  under  the  influence  of 
something  present  in  the  pancreas  which  can  be  extracted 
from  it  by  boiling  water  and  is  soluble  in  alcohol.  Neither  the 
muscle  juice  nor  the  alcoholic  extract  of  pancreas  has  any 

1  Maze,  A.  P.  /.,  18,  378  ;  Portier,  id.  633,  1904  ;  Cohnheim,  H.-S.  Z. 
39,  348,  1903  ;  Arnheim  and  Rosenbaum,  ff.-S.  Z.  40,  233,  1903. 


HI.]  THE  PANCREAS  AND  GLYCOLYSIS  67 

glycolytic  action  by  itself,  but  the  two  together,  provided  there 
is  not  an  excess  of  the  latter,  produce  a  powerful  action.  The 
nature  of  the  substances  formed  from  the  sugar  in  his  experi- 
ments he  has  not  determined.  Results  in  some  respects  similar 
to  Cohnheim's  have  been  obtained  by  Rachel  Hirsch.1 

But  here,  too,  there  are  considerable  difficulties  in  definitely 
adopting  the  scheme  of  correlated  interactions  of  different  parts 
of  the  body  as  laid  down  by  Cohnheim.  For  the  difficulty  of 
eliminating  bacterial  growth  and  preserving  at  the  same  time 
the  conditions  necessary  for  the  development  of  the  reactions 
described,  has  in  the  hands  of  others  so  far  proved  insuperable.2 
Till  these  conditions  are  more  clearly  defined,  and  it  becomes 
possible  to  control  them  more  completely  than  at  present  is 
the  case,  we  cannot  be  certain  that  such  experiments  tell  us 
anything  about  reactions  due  to  the  cell- substances  themselves, 
and  consequently  those  which  are  carried  out  in  the  living 
body. 

Glycolysis,  whether  in  the  blood  or  in  the  tissues,  is  a  subject 
therefore  on  which  it  is  possible  for  the  present  to  make  but 
few  positive  statements :  neither  the  conditions  under  which 
it  occurs,  nor  the  nature  of  the  change  involved,  has  been 
determined  in  such  a  way  as  to  give  final  and  conclusive 
results. 

Occasion  arose  in  the  course  of  the  last  lecture  to  refer  to  one 
way  in  which  some  of  the  sugar  in  the  body  is  believed  to  be 
broken  down.  We  then  saw  that  glycuronic  acid,  which  differs 
from  glucose  only  in  having  a  carboxyl  group  substituted  for 
the  terminal  primary  alcohol  group  of  the  sugar  molecule,  had 
been  proved  by  P.  Mayer  to  be  formed  from  glucose  in  the 
rabbit.  This  substance  occurs  in  traces  in  normal  human  urine, 
about  half  a  decigramme  daily,  combined  for  the  most  part  with 

1  R.  Hirsch,  H.  B.  4,  535,  1903. 

2  Embden  and  Glaus,  H.  B.  6,  p.  214,  1905.     Harden  and  Young  have 
repeated  Cohnheim's  experiments,  with  the  one  modification  that  the  muscle 
was  ground  with  sand,  instead  of  being  frozen  and  cut  in  shavings  with 
Kossel's  apparatus,  as  done  by  Cohnheim.     But  they  found  no  trace  of  the 
action  described.     (Private  communication.} 


68  CARBOHYDRATE  CATABOLISM  [LECT. 

phenol : l  it  is  also  present  in  bile,2  in  the  blood,3  and  in  faeces. 
But  the  administration  of  camphor  or  chloral  causes  it  to  appear, 
combined  with  these  substances,  in  the  urine  in  considerable 
quantities.  This  fact  may  be  taken  to  indicate  that  it  is 
formed  as  an  intermediate  product  in  metabolism,  possibly  in 
still  larger  quantities,  and  that  it  does  not  come  into  evidence 
unless  it  happens  to  be  caught  and  fixed  by  combination  with 
such  a  substance  as  phenol,  because  it  is  otherwise  completely 
oxidised.  Mayer  found  in  rabbits  no  indication  that  doses  of 
glycuronic  acid  up  to  about  5  g.  were  not  as  completely  oxidised 
as  glucose  itself.  When  larger  amounts  were  given,  however, 
the  acid  was  found  in  the  urine,  partly  free  and  partly  combined 
with  the  phenol  that  would  otherwise  have  been  present 
combined  with  sulphuric  acid.  There  was  no  increase  in  the 
phenol,  but  more  of  it  was  paired  with  glycuronic  acid  and  less 
with  sulphuric  acid.4  The  fact  that  normally  only  a  small 
fraction  of  the  phenol  pairs  with  glycuronic  acid  may  be 
taken  to  indicate  that  glycuronic  acid  does  not  occur 
in  the  body  normally  in  such  large  quantities  as  in  these 
experiments,  even  as  an  intermediate  product  of  metabolism, 
and  therefore  that  the  whole  of  the  sugar  oxidised  in  the  body 
does  not  go  through  the  changes  which  in  the  first  instance 
give  rise  to  the  formation  of  this  acid.  But  the  absorption  of 
glycuronic  acid  from  the  intestine,  it  must  be  remembered,  is 
not  the  same  as  the  formation  of  glycuronic  acid  in  the  cells  in 
the  course  of  oxidation  of  sugar.  Stress  cannot  always  be  laid 
on  arguments  from  the  results  of  administering  intermediate 
products  of  metabolism,  but  it  is  remarkable  that  doses  of  from 
15  to  25  g.  of  glycuronic  acid  proved  fatal  to  rabbits,  and  in 
such  animals  considerable  quantities  of  oxalic  acid  were  found 
in  the  liver,  60  to  80  mg.  instead  of  mere  traces.  All  that  we 
can  say,  therefore,  is  that  it  does  not  appear  probable  that  the 
normal  course  of  sugar  metabolism  necessarily  and  always 

1  Mayer  and  Neuberg,  H.-S.  Z.  29,  267,  1900. 

2  Bial,  H.-S.  Z.  45,  263,  1905. 

3  Mayer,  H.-S.  Z.  32,  518,  1901. 

4  Mayer,  Z.f.  k.  M.  47,  68,  1902. 


in.]  GLYCURONIC  ACID  AND  XYLOSE  69 

involves  the  formation  of  glycuronic  acid,  though  clearly  some 
of  the  sugar  is  oxidised  in  this  way. 

It  has,  however,  even  been  contended  that  this  substance  is 
not  derived  from  sugar  at  all.  Loewi  concludes,  from  his 
experiments  on  the  administration  of  camphor  to  dogs  in 
phlorrhizine  diabetes,  that,  at  any  rate  under  these  conditions, 
it  is  not.  In  one  case  a  dose  of  10  g.  of  camphor,  which  would 
and  apparently  did  combine  with  as  much  glycuronic  acid  as 
could  be  formed  from  12  g.  of  .sugar,  instead  of  diminishing  the 
amount  of  sugar  excreted  to  this  extent,  was  accompanied 
actually  by  an  increase  of  the  sugar  excreted,  amounting  to 
about  20  g.,  or  nearly  30  per  cent,  of  the  average  amount 
excreted  on  the  previous  days.  In  the  three  other  experiments, 
in  each  of  which  two  doses  of  camphor  were  given,  on  four 
occasions  the  sugar  excretion  was  diminished,  on  two 
unchanged.1  The  results,  therefore,  varied  somewhat  widely, 
and  it  is  questionable  whether  they  furnish  proof  that  in  the  dog 
under  these  conditions  the  glycuronic  acid  is  not  formed  from 
carbohydrates.  If  it  is  possible  for  sugar  to  be  synthesised  in 
the  body  from  material  derived  from  either  proteids  or  fats, 
then  clearly  glycuronic  acid  may  be  remotely  connected  with 
either  of  these  substances,  though  directly  formed  from  sugar. 

The  probability  that  the  xylose  found  in  nucleic  acids  may 
be  the  next  product  after  glycuronic  acid  in  the  series  starting 
from  glucose  along  this  line  of  catabolic  change,  has  also  been 
referred  to  in  the  last  lecture.  But  beyond  this  possibility  we 
cannot  trace  the  catabolism  of  sugar  in  this  direction  at  all. 

In  connection  with  the  origin  of  xylose  we  have  to  take  into 
account  the  phenomena  of  pentosuria.  This  remarkable 
anomaly  of  metabolism — it  does  not  amount  to  a  disease,  since 
it  is  not  accompanied  necessarily  by  any  disturbance  of  health — 
was  discovered 2  before  it  was  known  that  pentoses  entered  into 
the  composition  of  any  substances  in  the  body.  When  this 
came  to  be  known,  there  was  not  unnaturally  a  tendency  to 
associate  the  pentose  in  pentosuria  with  the  pentose  in  the 

1  O.  Loewi,  S.  A.  47,  56,  1902. 

2  Salkowski  and  Jastrowitz,  Cbl.  Med,  Wiss^  Nos.  19  and  35,  1892. 


70  CARBOHYDRATE  CATABOLISM  [LECT. 

nucleic  acids.  But  with  the  determination  of  the  exact  nature 
of  these  pentoses,  the  identification  of  active  /-xylose  in  the 
nucleic  acids  from  the  pancreas  and  liver,1  and  of  inactive 
arabinose  in  the  urine,2  this  tendency  was  found  to  lead  to 
difficulties.  The  sugar  in  the  nucleic  acids  is  represented  by 
the  formula — 

OH     H         OH 
C 


COH.C     .     C    .     C     .     CH2OH 
>H     H 


The  sugar  in  the  urine  in  pentosuria  is  the  inactive  mixture  of 
H         OH     OH 

COH.C     .     C     .     C     .     CH2OH          (rf-Arabinose) 

OH      H         H 
and 

OH     H        H 

I  I  I 

COH.C     .     C     .     C     .     CH2OH         (/-Arabinose). 

H         OH      OH 

The   second    of    these   (/-arabinose)   is    related  to  /-xylose  as 
galactose  is  to  glucose. 

If,  therefore,  it  were  the  optically  active  /-arabinose  alone 
that  were  found  in  the  urine  in  these  cases,  we  might  explain 
its  presence  either  by  a  stereoisomeric  transformation  of  /-xylose 
similar  to  that  which  has  been  suggested  to  account  for  the 
formation  of  galactose  from  glucose,  or  by  deriving  it  from 
active  galactose,  as  xylose  has  been  derived  from  glucose.  But 
apart  from  the  fact  that  neither  by  feeding  dogs  on  large 
quantities  of  pancreas  could  pentosuria  be  induced,  nor  in  the 
subjects  of  pentosuria  could  any  relationship  be  observed 
between  the  pentose  excreted  and  the  nature  or  amount  of  the 

1  Neuberg,  B.  35,  1467,  1902  ;  and,  Wohlgemuth,  H.-S.  Z.  37,  475,  1903. 

2  Neuberg,  B.  33,  2243,  19°°- 


in.]  PENTOSURIA  71 

carbohydrates  taken  as  food,1  it  would  be  contrary  to  all  that  is 
known  concerning  optically  active  compounds  to  suppose  that 
inactive  arabinose  should  be  formed  directly  from  either  active 
xylose  or  active  galactose.  But  if  the  terminal  carbon  atoms  of 
galactose  undergo  any  change  by  which  the  groups  into  which 
they  enter  become  indistinguishable ;  if,  for  instance,  both  are 
oxidised  to  carboxyl,  giving  rise  to  mucic  acid,  or  if,  by 
reduction  of  the  aldehyde  group  both  become  primary  alcohol 
groups,  giving  dulcite, 

COOHf  OH       H        H       OH  \     COOH 

or      \     C     .     C     .     C     .     C     I        or 
CH2OHl    H      OH      OH       H    J    CH2OH 

the  substances  formed  are,  owing  to  the  symmetrical  disposition 
of  the  asymmetric  carbon  atoms  in  the  middle  of  the  molecule, 
optically  inactive.  If,  then,  a  derivative  of  galactose  fulfilling 
this  condition  by  subsequent  changes  gave  rise  to  arabinose,  as 
Neuberg  suggests,2  both  modifications  must  occur  equally 
readily  and  the  arabinose  be  inactive.  For  instance,  if  carbonic 
acid  were  removed  from  one  end  of  mucic  acid,  it  must  be 
equally  easily  removed  from  either  end,  and  the  resulting 
arabonic  acid  must  be  inactive,  and  on  reduction  give  inactive 
arabinose.  The  occurrence  of  inactive  arabinose  as  a  product 
of  animal  metabolism  is  so  remarkable  a  phenomenon,  that  it  is 
important  to  be  alive  to  its  significance  and  its  bearings  on  the 
metabolism  of  carbohydrates  in  general. 

1  Bial  and  Blumenthal,  D.  M.  W.,  No.  22,  1901. 
*2  Neuberg,  Ergeb.,  iii.,  429. 


LECTURE    IV 

THE  ASSIMILATION   AND   SYNTHESIS   OF   FAT 

THE  assimilation  of  carbohydrates  involves  in  all  cases  some 
change  in  the  nature  of  the  carbohydrates.  Starch  and  sugars 
are  converted  into  hexoses  ;  the  hexoses  undergo  certain  trans- 
formations, and  are  built  up  into  glycogen.  But  it  may  possibly 
be  questioned  whether  carbohydrates  are  synthesised  under 
ordinary  circumstances  from  material  which  is  not  carbohydrate 
in  nature  to  start  with.  The  synthesis  of  sugar  can  certainly  be 
effected  in  animals,  but  we  do  not  yet  certainly  know  that  such 
a  synthesis  commonly  takes  place  in  normal  metabolism,  nor  do 
we  know  certainly  what  is  the  exact  nature  of  the  chemical 
combinations  made  use  of  in  this  synthesis,  when  it  occurs. 

In  the  metabolism  of  the  fats  the  situation  is  different. 
There  is  no  doubt — it  was  proved  fifty  years  ago — that  fats  are 
built  up  from  material  of  quite  a  different  nature,  in  animals 
that  are  in  every  respect  normal.  On  the  other  hand,  all  the 
evidence  points  to  fats  being  taken  up  and  assimilated  without 
transformation.  The  glycerine  and  fatty  acids  are,  it  is  true, 
temporarily  dissociated  in  digestion  for  the  purpose  of  absorp- 
tion ;  but  they  are  recombined  almost  immediately  without  any 
other  change,  and  they  are  found  stored  up,  when  not  required 
for  immediate  use,  in  the  identical  form  in  which  they  were 
taken  in  the  food.  Thus  it  is  familiar  that  the  nature  of  the  fat 
in  an  animal's  body  largely  depends  on  the  nature  of  the  fat  in 
its  food.  In  certain  vegetable  oils  fatty  acids  occur  as  glycer- 

ides  which  are  not  commonly  found  in  animal  fats.     Colza  oil 

73 


LECT.  iv.]    FAT  IS  ASSIMILATED  WITHOUT  CHANGE          73 

or  rape-seed  oil,  for  instance,  contains  in  large  quantities  the 
glyceride  of  an  acid  of  the  oleic  series,  erucic  acid,  C22H42O2 ;  and 
linseed  oil  is  composed  largely  of  linolein,  linoleic  acid  being  a 
derivative  of  stearic  acid,  with  unsaturated  linkages  at  two  points 
in  the  chain,  and  having  the  formula  C18H32O2.  If  either  of 
these  oils  is  made  use  of  as  food,  or  if  fats  even  in  which 
the  unsaturated  carbon  atoms  have  been  saturated  with  the 
halogens  iodine  or  chlorine  are  taken,  then  these  unusual  glycer- 
ides  are  laid  down  in  the  connective  tissues  without  change, 
just  as  if  it  were  perfectly  normal  for  them  to  be  there.  Even 
in  man  it  is  known  that,  if  erucic  acid  be  given  by  the  mouth, 
this  acid,  as  glyceride,  but  otherwise  unchanged,  is  found  in  the 
chyle.  A  dog's  fat  is  usually  so  compounded  that  it  can  absorb, 
by  reason  of  the  amount  of  olein  that  it  contains,  from  51  to  56 
per  cent,  of  its  weight  of  iodine.  But  if  a  dog,  after  a  period  of 
starvation  in  which  most  of  its  own  fat  is  consumed,  is  fattened 
on  fat  mutton,  the  fat  now  found  in  its  connective  tissues  absorbs 
only  about  42  per  cent,  of  iodine.  It  is  almost  as  poor  in  olein 
as  mutton  fat  itself,  which  absorbs  only  about  36  per  cent,  of 
iodine.  One  form  of  fat  appears  therefore  to  serve  as  well  as 
another,  if  only  it  is  absorbed.  So  that  the  assimilation  of  fat  is 
a  subject  on  which  there  is  not  much  to  be  said.  The  character 
of  the  fat  in  the  body  is  determined  primarily  by  the  nature  of 
the  fat  in  the  food ;  in  part,  too,  doubtless  by  the  nature  of  the 
fat  synthesised  from  substances  other  than  fat  within  the  body, 
and  also  in  part  by  the  ease  with  which  the  different  kinds  of 
fat  are  absorbed.  Fats  are  absorbed  with  difficulty  if  their 
melting  point  is  higher  than  the  body  temperature.  Olein  is  in 
all  animals,  but  especially  in  cold-blooded  animals  such  as  fish, 
much  more  easily  absorbed  than  fats  with  a  high  melting 
point,  such  as  palmitin  and  stearin.  But  whatever  fat  is 
absorbed  is  ip so  facto  assimilated.  No  transformation  is  neces- 
sary, for  all  the  fatty  acids  are  equally  adapted  to  the 
reactions  by  which  their  chemical  energy  is  subsequently 
liberated  in  the  body. 

In  the  synthetic  metabolism  of  fats,  therefore,  we  need  not 
stop  over  problems  of  assimilation.     It  is  the  actual  building  up 


74  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT. 

of  fat  from  substances  of  a  different  kind,  that  furnishes  us  with 
all  our  problems  in  this  division  of  our  subject. 

The  fats  are  glycerine  esters  of  higher  fatty  acids.  It  is 
necessary  therefore  to  give  what  account  we  can  of  the  origin  of 
the  fatty  acids,  of  the  origin  of  the  glycerine,  and  of  the  process 
by  which  these  are  combined. 

The  last  of  these  three  subjects,  to  begin  with  the  one  that 
presents  least  difficulty,  has  not  failed  to  attract  its  full  share  of 
the  attention  of  physiologists.  The  union  of  glycerine  and 
higher  fatty  acids  can  be  effected  in  the  laboratory  by  heating 
the  reagents  together  to  high  temperatures.  The  reverse 
process,  saponification  of  the  glycerides,  can  be  brought  about 
by  the  use  of  alkalies,  especially  in  the  presence  of  alcohol,  or  by 
steam  at  300°  C.  In  the  body  both  saponification  and  the 
reverse  operation  are  familiar,  and  apparently  both  are  effected 
by  one  and  the  same  means.  The  reaction,  a  reversible  one,  is 
the  work  of  an  enzyme  or  enzymes  which  are  found  widely 
distributed  in  the  body  ;  in  the  pancreas  and  its  secretion,  in  the 
stomach  and  its  secretion,  in  the  liver,  the  mamma,  the  intestine, 
the  connective  tissues,  and  perhaps  in  the  blood.  A  lipase  has 
been  shown  to  be  present  in  all  these  tissues,  which  saponifies 
not  only  fats,  but  other  esters  like  ethyl  butyrate.  Aqueous 
extracts  of  these  organs  treated  with  this  ester  in  the  presence 
of  toluene  develop  an  acid  reaction,  while  controls  to  which  no 
ester  is  added  do  so  to  a  comparatively  slight  degree,  and  other 
controls  which  are  boiled  remain  unchanged :  treated  with 
butyric  acid  and  ethyl  alcohol,  they  bring  about  the  synthesis  of 
the  ester,  to  judge  at  least  by  the  smell.1  Hanriot  found  that 
lipase  causes  the  combination  of  butyric  acid  and  glycerine,  and 
obtained  monobutyrin  in  this  way  ;  since  it  can  also  saponify 
esters  in  which  the  alcohol  and  acid  may  be  of  widely  different 
characters,  its  action  is  perhaps  a  general  one,  consisting  in  the 
acceleration  of  the  approach  to  the  equilibrium  point  in  the 
system,  acid  +  alcohol  ^  ^  ester  +  water.  The  significance  of 
this  action  in  physiology  appears  to  be  this :  when  fat  is  to  be 

1  Kastle  and  Loewenhart,  Am.  Ch.  Jl.  24,  491,  1900 ;  and,  Loewenhart, 
Am.JL  Phys.  vi.,  331,  1902. 


iv.]  ACTION  OF  LIPASE  75 

transferred  across  certain  cell  membranes,  it  is  saponified ;  when 
it  is  to  come  to  rest,  the  ester  is  formed.  Under  certain 
conditions  this  reaction  proceeds  in  the  one  direction ;  under 
others,  in  the  other.  Thus,  in  digestion  saponification  of  fats 
precedes  their  entry  into  the  epithelium  ;  in  starvation,  it  enables 
the  connective  tissue  fat  to  leave  the  fat  cells,  and  so  reach  the 
circulation,  to  be  distributed  to  the  organs  that  require  chemical 
energy  with  which  to  do  their  work. 

Exactly  how  and  why  saponification  aids  the  passage  of 
fats  through  cell  membranes  is  not  known.  The  fatty  acids  are 
no  more  soluble  in  water  than  the  fats  themselves,  and  their 
alkaline  salts  have  been  shown  to  be  poisonous  when  injected 
into  the  circulation — 0.15  g.  of  sodium  oleate  per  kg.  or 
less  of  the  palmitate  or  stearate  is  fatal  for  rabbits.1  They 
cause  a  great  fall  of  blood  pressure,  and  then  diastolic  arrest  of 
the  heart  and  loss  of  coagulability  of  the  blood.  But  the  free 
fatty  acids  though  insoluble  in  water  are  soluble  in  certain 
fluids  found  in  the  body ;  in  the  bile,  for  instance,  and  still 
more  in  the  fluid  found  in  the  intestine;2  and  it  is  probable 
that  it  is  as  fatty  acid  dissolved  in  some  unknown  agent  in 
the  body  fluids  that  the  fats  are  transferred  through  cell 
membranes. 

Neither  is  it  easy  to  form  a  conception  of  the  conditions 
which  determine  the  direction  in  which  this  reversible  reaction 
is  to  take  place :  how  it  is,  for  instance,  that  during  inanition 
the  stored  fat  is  so  easily  put  into  circulation,  and  in  ordinary 
conditions  of  nutrition  is  left  at  rest  ?  If  it  is  the  enzyme  in 
the  connective  tissues  that  brings  about  the  synthesis  and 
deposition  of  the  glycerides,  how  it  is  that  this  synthesis 
continues  to  be  carried  out  even  when  the  cells  are  already 
loaded  up  with  fat  ? 

If  it  is  right  to  suppose  that  this  widely  distributed  enzyme, 
lipase,  serves  the  purpose  of  setting  in  motion  changes  in  the 
distribution  of  fat  in  the  body,  it  is  clear  that  its  function  is 
important.  But  the  change  it  effects  is  isothermic.  One 

1  Munck,  D.  R.  A.,  supp.,  p.  117,  1890. 

2  Moore  and  Rockwood, //.  of  Phys.  21,  58,  1897. 


76  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT. 

gramme-molecule  of  ethyl  butyrate  gives,  on  combustion, 
851.3  Cal. 

I  g.  mol.  of  Ethyl  alcohol  gives     .     325.7  Cal. 
I  g.  mol.  of  Butyric  acid  gives       .     524.4  Cal. 

850.1 
Similarly, 

3  g.  mols.  of  Stearic  acid  on  combustion  give   .     8017  Cal. 
i  g.  mol.  of  Glycerine  gives      .     .    »         .         .       396  Cal. 

8413 

and  I  g.  mol.  of  stearin  (i  g.  =  9.43  Cal.)  would  give  8393  Cal. 
The  differences  fall  within  the  limits  of  experimental  error. 
In  whichever  direction,  therefore,  a  lipolytic  enzyme  acts,  it 
neither  adds  to  nor  deducts  from  the  fund  of  energy  on  which 
the  organism  has  to  draw.  In  the  complex  of  chemical  changes 
that  constitute  the  life  of  an  organism,  there  are  many  which, 
though  of  service,  are  not  intimately  associated  with  the 
phenomena  of  its  life.  It  may  be  of  service  in  digestion  that 
the  free  hydrochloric  acid  of  the  gastric  juice  should  be 
neutralised  in  the  duodenum  ;  but  such  a  reaction  is  not  one  of 
peculiarly  physiological  moment  in  the  sense  in  which  the 
liberation  of  the  chemical  energy  of  sugars  or  fats  is,  when  with 
that  energy  the  heart,  for  instance,  is  enabled  to  do  its  work. 
The  saponification  of  fat,  or  the  reverse  process,  is  not  one  of 
the  changes  in  which  a  vital  transformation  of  energy  is  brought 
about.  The  fatty  acids  are  at  one  moment  combined  with 
glycerine,  at  the  next  they  are  free :  it  makes  no  difference  how 
many  times  this  to-and-fro  movement  is  executed,  the  groups 
concerned  are  in  either  form  equally  charged  with  what  they 
are  to  contribute  to  the  life  of  the  organism. 

But  the  synthesis  of  the  high  fatty  acids  from  the  food-stuffs 
other  than  fat  is  a  chemical  change  so  peculiarly  characteristic 
of  living  matter  that  its  importance  is  of  a  different  order. 
That  carbohydrates  can  be  converted  into  fat  at  all,  we  know 
only  from  the  physiological  study  of  animal  metabolism.  It  is 
a  change  for  which  living  organisms  have  the  monopoly.  The 


iv.]  LAWES  AND  GILBERTS  EXPERIMENTS  77 

earliest  method  of  studying  metabolism,  what  is  sometimes 
called  the  balance-sheet  method,  which  consists  in  the  analysis 
of  the  food  on  the  one  hand  and  the  excretory  output  from  the 
body  on  the  other,  as  well  as  sometimes  the  whole  body  of  the 
animals  employed,  and  then  balancing  the  elementary  items,  has 
rendered  no  greater  service  to  physiology  than  this.  Lawes 
and  Gilbert's  experiments  on  the  fattening  of  farm  stock,  carried 
out  on  the  experimental  farm  at  Rothamsted,  nearly  fifty 
years  ago,  were  the  first  of  a  large  number  of  similar  experi- 
ments, which  all  prove  what  was  first  suggested  by  Liebig,  that 
large  quantities  of  fat  are  built  up  in  animals  from  the  carbo- 
hydrates of  the  food.  Now  these  fats  contain  about  96  per  cent, 
of  their  weight  of  the  higher  fatty  acids,  long  normal  chains, 
that  is,  of  1 6  or  1 8  carbon  atoms,  saturated  or  almost  saturated 
with  hydrogen,  and,  except  for  the  2  oxygen  atoms  of  the  acid- 
carboxyl  group,  with  hydrogen  alone ;  while  in  the  sugars,  the 
chains  of  6  carbon  atoms  are  oxidised  all  along  the  line.  The 
change  is  remarkable  however  it  is  to  be  looked  at.  Supposing 
for  a  moment  that  3  sugar  molecules  could  join  up  end  to  end 
in  a  normal  chain,  no  less  than  16  out  of  the  18  oxygen  atoms 
must  be  dislodged  for  it  to  become  stearic  acid,  and  those  that 
remain  be  rearranged.  And  the  more  we  call  to  our  aid  of 
what  synthetic  chemistry  has  to  teach,  the  more  remarkable 
the  change  appears  ;  so  that  for  the  chemist,  the  only  solution  of 
the  problem  has  often  appeared  to  be  to  regard  the  whole 
matter  as  a  physiological  fiction.  At  any  rate,  the  fifty  years 
since  it  was  first  proved,  which  have  been  more  eventful  and 
fruitful  in  the  history  of  chemistry  than  any  similar  period  has 
perhaps  ever  been  in  the  history  of  any  other  science,  have 
hardly  produced  a  serious  attempt  to  solve  the  problem. 

The  problem  gains  in  interest,  too,  if  we  take  into  account 
the  fact  that  fats  and  oils  are  most  probably  synthesised  from 
carbohydrates  wherever  they  are  synthesised  in  Nature. 

Whatever  the  solution  may  prove  to  be,  there  are  certain 
facts,  for  the  most  part  very  familiar,  which  can  hardly  fail  to  be 
of  significance  as  clues  to  its  elucidation.  Of  the  fatty  acids  that 
occur  in  Nature,  those  that  contain  more  than  5  carbon  atoms 


78  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LKCT. 

are,  with  hardly  an  exception,  the  members  of  the  series  which 
contain  an  even  number  of  carbon  atoms.1  Of  these  even 
number  acids  the  16  and  18  carbon  acids  are  much  the  most 
abundant,  but,  nevertheless,  all  the  other  intermediate  members 
are  found,  in  traces  at  any  rate.  The  connective-tissue  fat,  it  is 
true,  does  not  contain  these  lower  acids,  but  that  is  fat  which  is 
out  of  the  current  of  metabolism  passively  waiting  for  its  time 
to  come.  But  in  milk,  besides  comparatively  large  quantities 
of  the  glyceride  of  butyric  acid,  caproin,  caprylin,  caprin,  laurin, 
and  myristin  are  all  found,  in  traces  at  least,  the  glycerides,  that 
is,  of  the  acids  with  6,  8,  10,  12,  and  14  carbon  atoms.  And 
in  all  cases  these  acids  are  the  normal  acids,  with  straight  un- 
branching  chains.  Then  in  addition  to  these  normal  saturated 
acids  certain  unsaturated  acids  occur,  principally  oleic  acid,  but 
also,  in  plants  at  any  rate,  others,  some  of  which  are  unsaturated 
in  more  than  one  linkage  of  the  chain.  And  even  in  animals 
the  twice  unsaturated  acid,  linoleic  acid,  C1SH32O2,  is  said 2  to 
account  for  25  per  cent,  of  the  fatty  acids  in  the  lecithine  of  the 
hen's  egg,  and  also  is  believed  to  be  present  in  lard  and  other 
animal  fats :  for  instance,  the  fat  of  the  hare  and  the  horse,  in 
the  latter  of  which  it  amounts  to  about  10  per  cent  of  all  the 
fatty  acids.  In  plants,  too,  besides  unsaturated  acids,  at  any 
rate  one  oxy-acid,  ricinoleic,  or  hydroxyoleic  acid,  occurs 
abundantly  in  castor  oil ;  and  other  oxy-acids  are  said  to  occur 
in  the  wool  fat  of  animals. 

It  is  possible  that  these  facts  should  all  be  associated 
together,  and  that  there  is  some  genetic  relationship  between 
these  several  forms  in  which  fatty  acids  occur  ;  that  they  indicate, 
in  fact,  steps  in  the  process  by  which  all  alike  are  formed — a 
process  which  tends  to  be  completed,  and  result  in  the  evolution 
of  the  highest  members  of  the  group,  those  which  actually  occur 
in  the  largest  quantities.  If  there  is  no  such  relationship,  then 
each  fatty  acid  will  present  a  problem  of  its  own,  and  our  diffi- 

1  Belief  in  the  occurrence  in  nature  of  the  acid  C1-7H34O2,  margaric  acid, 
has  been  of  late  resuscitated  from  time  to  time,  but  apparently  on  insuffi- 
cient and  mistaken  evidence. — v.  Holde,  B.  38,  1247,  1905. 

2  Henriques  and  Hansen,  Sk.  A.  14,  390,  1903. 


iv.]  THE  GENESIS  OF  HIGHER  FATTY  ACIDS  79 

culties  will  be  the  greater.  Thus  the  occurrence  of  palmitic 
acid  side  by  side  with  stearic  makes  it  improbable  that  the 
1 8  carbon  atoms  of  stearic  acid  are  joined  up  in  groups  of  6  at 
a  time.  And  the  occurrence  of  all  the  even  number  acid  makes 
it  probable  that  the  chains  are  built  up  by  the  addition  of  2 
carbon  atoms  at  a  time.  If  this  conception  can  be  combined 
with  one  which  will  account  for  the  appearance  on  occasions  of 
oxy-acids  and  of  unsaturated  linkages  in  the  chain,  then  the 
problem  of  the  building  up  of  fatty  acids  will  begin  to  present 
a  comparatively  simple  aspect,  such  as  it  is  probable  that  the 
natural  process  has.  Since  all  fatty  acids  alike  are  equally 
available  for  catabolic  processes,  it  is  probable  that  the  reactions 
on  the  down-grade  of  fat  metabolism  are  not  specific  for  each 
fatty  acid  ;  that  palmitic  acid,  for  instance,  because  it  contains 
1 6  atoms  of  carbon,  does  not  break  down  differently  from 
stearic  acid  with  18.  The  reactions  in  the  catabolism  of  fat 
are  such  as  to  apply  equally  to  them  all.  The  reactions  by 
which  they  are  generated  are  similarly  likely  to  be  the  same 
for  all. 

Nearly  thirty  years  ago,  in  a  series  of  papers  on  the  changes 
effected  by  micro-organisms  (Gahrungs-processe)  Hoppe-Seyler 
drew  attention  to  the  similarity  between  many  of  these  changes 
and  those  brought  about  by  caustic  alkalies.1  And  in  particular 
he  devoted  especial  study  to  the  fate  of  lactic  acid  under  these 
influences.  He  found  that  lactic  acid  heated  with  caustic 
alkalies  to  a  temperature  of  220°  C.  began  to  give  off  hydrogen  : 
as  the  temperature  was  slowly  raised,  the  reaction  became  at 
first  more  energetic,  then  gradually  slowed  down,  and  finally 
stopped  at  about  300°  C.  The  fused  mass,  dissolved  in  water 
and  acidified  with  sulphuric  acid,  gave  up,  besides  carbonic  acid, 
upon  distillation  considerable  quantities  of  fatty  acids,  principally 
acetic  and  butyric,  but  also  caproic  acid.  In  addition  to  these 
volatile  acids,  however,  he  always  found  small  quantities  of  solid 
insoluble  acids  which  melted  in  hot  water  to  form  oily  drops  : 
their  alkaline  salts  were  soluble  in  water  or  alcohol,  and  the 
aqueous  solutions  of  these  salts  when  concentrated  and  cooled 
1  Hoppe-Seyler,  ff.-S.  Z.  3,  351,  1879. 


80  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LEG*. 

set  to  a  jelly,  as  a  soap  solution  does.  The  melting  point  of  the 
acids  he  found  to  vary  in  different  experiments,  but  it  was 
always  lower  than  that  of  palmitic  acid,  and  their  molecular 
weight  also  varied,  and  was  lower  than  that  of  palmitic  acid.  He 
concluded  that  these  insoluble  acids  were  mixtures  of  higher 
even  number  members  of  the  acetic  series  formed  from  the 
lactic  acid  in  order,  in  diminishing  quantities  as  the  chains 
became  longer,  and  by  the  same  reaction  as  that  by  which  the 
butyric  and  caproic  acid  were  formed. 

Pasteur,  and  also  Fitz,  had  previously  described  the  forma- 
tion of  butyric  and  caproic  acid  from  lactic  acid  by  bacteria,  and 
Hoppe-Seyler  looks  to  these  reactions  as  the  simplest  form 
of  the  change  of  such  great  importance  in  the  physiology  of 
plants  and  animals  alike  by  which  carbohydrates  are  converted 
into  fats. 

On  repeating  these  experiments  of  Hoppe-Seyler's  it  is  easy 
to  confirm  the  formation  of  volatile  fatty  acids,  and  also  to 
obtain  small  quantities  of  a  substance  or  substances  which  are 
not  volatile,  are  insoluble  in  water,  and  in  some  respects  behave 
like  higher  fatty  acids.  But  the  dark  oily  drops  have  a  tarry 
appearance,  and  if  separated  from  the  strong  salt  solution  in 
which  they  are  first  formed,  dissolved  in  alkali,  and  reprecipitated, 
they  tend  no  longer  to  float,  but  to  stick  to  the  sides  and 
bottom  of  the  vessel.  The  products  of  this  reaction  have  been 
recently  reinvestigated  by  Raper  at  the  Lister  Institute,  with 
the  result  that  besides  formic  and  acetic  acids  together  with 
butyric,  and  probably  caproic,  also  isobutyric  acid,  are  formed  : 
the  insoluble  acids,  described  as  higher  fatty  acids  by  Hoppe- 
Seyler,  are  of  low  molecular  weight,  are  unsaturated  compounds, 
since  they  decolorise  bromine  water  and  reduce  permanganate  in 
the  cold,  and  they  are  heavier  than  water.  Their  exact  nature 
was  not  determined,  but  they  are  clearly  not  the  higher  fatty 
acids,  such  as  palmitic  acid,  for  which  Hoppe-Seyler  took 
them.1 

Hoppe-Seyler's  experimental  support  for  his  conception  of 
the  manner  in  which  the  long  fatty  acid  chains  are  built  up,  goes 
1  Raper,//.  of  Phys.  32,  216,  1905. 


iv.]  HOPPE-SEYLER'S  EXPERIMENTS  81 

no  further,  therefore,  than  caproic  acid  at  any  rate.  But  the 
formation  of  even  butyric  and  caproic  acid  from  lactic  acid  must 
involve  a  synthesis,  and  just  such  a  synthesis  as  that  which 
occurs  in  Nature,  leading  to  a  series  of  acids  each  containing  two 
carbon  atoms  more  than  the  preceding  one.  The  reaction,  it  is 
true,  as  carried  out  by  him,  cannot  be  traced  beyond  the  second 
step,  but  if  it  were  continued  it  must  lead  to  the  other  even 
number  members  of  the  series.  It  is  possible  that,  though  mis- 
taken in  supposing  that  he  had  obtained  molecules  of  the  order 
of  magnitude  of  palmitic  and  stearic  acid,  he  was  really  on  the 
right  lines,  and  that  the  reaction  failed  to  reach  the  stage  which 
he  thought  it  reached,  only  because  it  was  diverted  by  the  inter- 
vention of  side  reactions :  a  selective  catalytic  action  might  be 
able  to  direct  and  keep  it  on  the  straight  course  by  accelerating 
just  those  phases  of  the  reaction  which  are  necessary,  and  by 
rapidly  carrying  it  past  the  points  where  side  reactions  are 
otherwise  apt  to  lead  it  astray. 

It  is  certainly  a  remarkable  coincidence  (if  only  a  coincidence), 
that  not  only  in  this  reaction  with  alkalies  caproic  acid  should 
appear  as  well  as  butyric,  but  that  in  the  butyric  fermentation  of 
sugar  by  micro-organisms,  caproic  acid  should  also  be  found 
among  the  products  so  regularly  that  commercial  caproic  acid  is 
obtained  from  crude  fermentation  butyric  acid.  It  seems  that 
lactic  acid  readily  gives  off  some  compound  containing  2 
carbon  atoms  that  tends  to  condense  with  itself,  once  to  form 
butyric  and  twice  to  form  caproic  acid. 

Exactly  how  the  groups  of  2  carbon  atoms  are  successively 
added  on,  even  if  the  reaction  goes  no  further  than  caproic  acid, 
we  can  of  course  only  speculate.  Hoppe-Seyler's  suggestion  as 
it  stands  (loc.  cit.,  p.  357)  hardly  commends  itself,  as  it  supposes 
a  tendency  in  ethyl  alcohol  to  condense  with  acetic  acid.  But 
since  lactic  acid,  as  is  well  known,  is  very  prone  under  the 
most  varied  treatment  to  split  into  aldehyde  and  formic 
acid,1  and  aldehyde  condenses  with  itself  very  readily,  there 
is,  for  the  present  at  any  rate,  more  to  be  said  for  such  a 
scheme  as  the  following,  which  is  based  on  a  suggestion 

i  Cf.  supra,  p.  53. 

F 


82 


ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT. 


made   in    the    first    instance    by    Nencki    and    developed    by 
Magnus  Levy.1 


(i)     2CH3. 


OH 


—  COOH   =   2  CH3 .  C 


+  2  H. COOH 


Lactic  acid  splitting  into  Aldehyde  and  Formic  acid. 

xH  /H  /H 

(2)     2CH3.C/      -   CH3.C<          .    CH2. 
^O  \OH 

Aldehyde  condensing  to  Aldol. 


(3)  + 

HO 
H 

H 

CH2  —  C 

=   CH3.CH2.CH2.COOH 
0 

OH 

\ 

OH 
H 

Aldol,  with  2  molecules  of  Water,  giving  Butyric  acid  and  Water. 

For  the  higher  members  of  the  series  similar  reactions  may 
account.  Aldehydes  with  6  carbon  atoms  have  been  obtained 
from  acetic  aldehyde  by  Riban  and  Kekule :  these  are  in  the 
one  case  a  twice  unsaturated  aldehyde,  and  in  the  other  an 
aldehyde  unsaturated  at  one  linkage  and  retaining  the  aldol 
or  oxy-form  in  the  other.  That  aldol  and  similarly  constituted 
bodies  readily  pass  into  unsaturated  compounds  by  the  loss  of 
water,  is  familiar ;  aldol  itself,  for  instance,  is  very  apt  to  become 
crotonic  aldehyde — 

CH9.CH   =   CH.C   ' 


Dialdane,  a  condensation  product  of  2  aldol  molecules,  was  first 
described  by  Wurtz,  who  showed  that  it  contained  8  carbon 
atoms.  He  prepared  the  acid  by  oxidation,  but  did  not  deter- 

1  ]Vt.  Levy,  D.  R.  A.,  p.  365,  1902, 


iv.]          ALDOL  CONDENSATION  AND  FATTY  ACIDS          83 

mine  whether  it  contained  a  branched  or  a  normal  chain,  nor 
whether  the  water  that  went  out  left  an  unsaturated  compound 
or  one  that  was  constructed  like  a  lactone,  though  he  assumed 
that  the  product  was  unsaturated,  and  therefore  had  the  formula 


XH  XH 

CH3.C<          .    CH0.CH   =    CH.C< 

\OH  X)H 


Raper  at  the  Lister  Institute  has  also  prepared  a  condensa- 
tion product  of  aldol.  When  examined  as  soon  after  isolation 
as  possible  by  the  cryoscopic  method,  this  had  approximately 
the  molecular  weight  of  a  dialdol.  The  molecular  weight  found 
was  183;  that  of  C8H16O4  is  176.  But  it  was  an  unstable  sub- 
stance tending  to  polymerise,  as  after  three  weeks  the  molecular 
weight  had  risen  to  278.  On  analysis,  it  showed  the  composi- 
tion of  the  substance  C8H14O3,  mixed  with  a  small  quantity  of 
C8H12O2.  The  iodine  absorptions  agreed  rather  with  the 
lactone  formula  than  with  that  of  an  unsaturated  compound, 
though  it  took  up  some  iodine.  In  order  to  determine  whether 
the  compound  contained  a  branched  or  a  normal  chain,  it  was 
first  converted  into  the  acid  (molecular  weight  from,  the  barium 
salt  1  68.8;  that  of  C8H14O4  being  174),  and  the  acid  was 
reduced  with  hydriodic  acid  in  the  way  in  which  Fischer  pre- 
pared heptylic  acid  from  mannose  carboxylic  acid.  The  acid 
was  distilled  in  steam,  and  from  the  sodium  salt  the  free  acid 
was  obtained,  but  on  distillation  it  underwent  decomposition  ; 
this  fact  also  probably  pointing  to  the  lactone  formula,  as  this, 
like  the  lactone  of  y-oxycaproic  acid,  is  not  reduced  by  hydriodic 
acid.  His  work  on  this  subject  has  had  to  be  interrupted,  but 
will,  he  hopes,  be  resumed  before  long,  and  in  the  meanwhile 
he  has  allowed  me  to  refer  to  it. 

The  aldol  condensation  is  a  general  reaction  of  aldehydes, 
and  it  is  not  impossible  that  conditions  may  occur  under  which,  by 
means  of  it,  the  fatty  acids  should  be  derived  in  series  from 
acetic  aldehyde.  It  is  true  that  condensation  products  of 
aldehyde  formed  under  ordinary  laboratory  conditions  contain 
mixtures  of  many  most  intractable  bodies,  and  that  each 


84  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT. 

aldehyde  as  it  is  produced  will  condense  in  this  way,  not 
only  with  itself,  but  with  every  other  aldehyde  present  in  the 
system ;  it  is  also  true  that  in  the  condensation  of  such  an 
aldehyde  as  butyric  aldehyde  with  acetic  aldehyde  in  vitro 
leads,  not  to  a  product  with  a  normal  chain,  but  to  one  in 
which  condensation  has  taken  place  at  the  a  carbon  atom, 
thus: 

/H          /H 
CH3.CH9.C/    —    C/ 

|  ^O 

CH3.C      —    H 

\>H 

so  that  at  present  it  seems  impossible  to  reproduce  the  con- 
ditions under  which  the  series  of  even  number  fatty  acids  can 
be  formed  by  aldol  condensation  from  acetic  aldehyde  as  the 
starting  point.  But  it  is  just  some  such  highly  reactive 
body  as  acetic  aldehyde,  condensing  under  the  guiding 
influence  of  some  catalytic  agent,  that  would  best  fit  the 
data  that  we  have  for  forming  a  conception  of  the  manner 
in  which  fats  are  produced  in  living  organisms  from  carbo- 
hydrates. 

Magnus  Levy  has  shown  that  during  aseptic  autolysis  of 
the  liver  the  strongly  acid  reaction  that  develops  is  due  to 
the  formation  of  lactic  acid,  acetic  acid,  and  butyric  acid.  His 
analyses  go  to  show  that  the  source  of  these  acids  is  probably 
mainly  carbohydrate,  and  he  regards  the  reactions  involved  as 
typical  of  those  by  which  the  higher  fatty  acids  are  formed 
from  carbohydrates.1  The  particular  acids  that  occur  are  sug- 
gestively in  favour  of  the  scheme  given  above,  by  which  the 
lactic  acid  in  giving  rise  to  acetic  aldehyde  would  lead  to  the 
formation  of  acetic  acid  by  oxidation,  and  of  butyric  acid,  by, 
in  the  first  place,  aldol  condensation,  and  then  by  changes  the 
net  result  of  which  is  to  transpose  the  /3-hydroxyl  and  the 
aldehyde  hydrogen. 

Supposing  that  the  synthesis  of  the  higher  fatty  acids  from 

1  Magnus  Levy,  H.  B.  2,  261,  1902. 


iv.]  NENCKI'S  HYPOTHESIS  85 

sugar  is  effected  on  these   lines,   then    the   energy   equations 
would  run  as  follows  :  — 

1  g.  mol.  Glucose,  *\  __    (2  g.  rnols.  Aldehyde  +  2  g.  mols.Formic  acid, 

677.2  Cal.        /         1          2x275.5  +  2x61.7  =  674.403!. 

2  g.  mols.  Aldehyde,]  f  i  g.  mol.  Aldol,)  f        i  g.  mol. 


or,  tracing  the  same  change  on  as  far  as  palmitic  acid  : 

4  g.  mols.  Glucose,)  f  i  g.mol.  Palmitic  acid  +  8  g.  mols.Formic  acid, 

2708  Cal.         I          I  2362  Cal.  +  494  Cal.  =  2856. 

In  the  first  stage  of  the  synthesis,  the  reaction  leading  to 
butyric  acid,  the  net  result  would  be,  supposing  the  formic  acid 
to  be  oxidised,  that  some  160  cal.,  or  nearly  25  per  cent,  of  the 
whole  energy  would  be  rendered  available  for  other  purposes. 
In  the  later  stages  leading  to  palmitic  acid,  some  of  the  energy 
derived  from  the  oxidation  of  the  formic  acid  would  be  required 
for  effecting  the  synthesis,  and  only  about  12.5  per  cent,  of  the 
original  amount  contained  in  the  sugar  would  be  set  free. 

What  the  organ  or  organs  in  the  body  are  that  effect  this 
change  of  sugar  into  fat,  we  have  not  the  means  of  deciding. 
In  Magnus  Levy's  experiments  butyric  acid  was  shown  to  be 
formed  in  the  liver,  and  probably,  as  he  himself  thinks,  from 
the  lactic  acid.  Those  livers  in  which  he  found  that  much 
butyric  and  acetic  acid  had  been  formed  contained  but  little 
lactic  acid,  and  vice  versa.  And  the  appearance  of  butyric  acid 
was  accompanied  by  the  evolution  of  hydrogen  and  carbonic 
acid,  just  as  it  is  in  butyric  fermentation,  whether  of  sugar  or 
of  lactic  acid.1  The  conversion  of  lactic  acid  into  butyric  acid, 
a  3  carbon  chain  into  one  of  4  carbon  atoms,  must  imply 
a  condensation.  And  Magnus  Levy's  experiments  suggest, 
as  he  points  out,  that  under  the  conditions  obtaining  in  the 

1  The  hydrogen  and  carbonic  acid  would  be  provided  for  in  the  scheme 
suggested  above  by  the  breaking  up  of  formic  acid  into  these  gases,  a  change 
that  occurs  in  vitro  at  160°  C.,  or  under  the  catalytic  influence  of  certain 
metals,  such  as  rhodium,  at  lower  temperatures. 


86  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT. 

body  the  liver  may  be  able  to  complete  the  series  of  condensa- 
tions, which  it  appears  to  begin  under  the  conditions  of  these 
experiments,  and  so  carry  out  the  synthesis  of  the  higher  fatty 
acids  also  from  lactic  acid. 

There  are  general  reasons,  too,  for  thinking  the  liver  to  be 
specially  concerned  in  this  process.  In  addition  to  its  situation 
on  the  path  of  absorption,  there  is  the  fact  that  the  change  in 
question,  however  it  be  brought  about,  is  one  that  involves 
energetic  reduction,  and  therefore  is  more  likely  to  be  feasible 
in  an  organ  where  the  larger  part  of  the  blood-supply  is  venous 
than  in  others  where  this  is  not  the  case. 

Siegert,  however,  found  that  there  was  no  change  in  the 
amount  of  fat  contained  in  pieces  of  liver  that  were  allowed  to 
undergo  aseptic  autolysis.1  Under  other  conditions,  the  total 
amount  of  higher  fatty  acids  contained  in  a  pulp  of  liver  cells 
after  incubation  in  a  current  of  air  has  sometimes  been  found 
to  be  greater  than  before,  and  still  greater  when  glycogen  was 
added.2  A  similar  change  has  been  observed  by  Hahn  in 
blood,  and  in  this  case  the  addition  of  sugar  made  the  difference 
more  marked.3 

Twenty  years  ago,  a  second  source  from  which  fat  was 
believed  to  be  derived  in  the  body  figured  more  prominently 
in  physiological  writings  than  the  carbohydrates.  It  was 
thought  that  proteids  were  converted  into  fat.  This  was 
mainly  due  to  Voit  and  Pettenkofer's  interpretation  of  their 
experiments,  in  which  dogs  were  fed  on  large  quantities  of  lean 
meat.  It  was  found  that  all  the  nitrogen  was  eliminated,  but 
not  all  the  carbon.  Since  the  amount  of  carbon  retained  in  the 
body  was  larger  than  could  be  accounted  for  as  glycogen,  they 
inferred  that  it  was  retained  and  stored  in  the  form  of  fat.  A 
dog,  for  instance,  was  given  2.5  kg.  of  lean  meat,  which  con- 
tained 85  g.  of  nitrogen,  and  according  to  their  reckoning  42  g. 
of  carbon  more  than  the  amount  excreted.  This  reckoning 
was  based  on  the  assumption  that  for  every  gramme  of  nitrogen 

1  Siegert,  H.  B.  i,  114,  1902. 

2  Hildesheim  and  Leathes,//.  of  Phys.  31,  1904. 

3  Hahn,  M.  M.  W.,  April  19,  1904. 


OF  THi 

UNIVERSITY 

OF 


VOIT'S  VIEW  OF  FAT  FORMATION  87 

in  lean  meat  there  were  3.68  g.  of  carbon,  whereas,  as  was 
pointed  out  by  Pfliiger  in  1891,  according  to  the  analyses  of 
Riibner,  this  figure  should  be  3.28.  For  every  gramme  of 
nitrogen,  therefore,  they  calculated  0.4  g.  of  carbon  too  much, 
giving  an  error,  with  85  g.  of  nitrogen,  amounting  to  34  g.  of 
carbon  at  one  stroke.  But  even  the  lower  figure  is  misleading. 
It  is  not  right  to  assume  that  the  whole  of  the  carbon  is  in 

C 

the  form  of  proteid.      What  the  ratio  of  —  in  the  proteid  of 

meat  really  is,  we  do  not  know.  For  myosin,  according  to 
Kiihne  and  Chittenden's  analyses,  the  value  of  this  ratio  is 
3.14.  But  myosin  is  by  no  means  the  only  proteid  in  meat; 
it  forms,  according  to  Danilewski,  less  than  half  of  the  whole 
proteid  of  meat,  and  there  are  proteids  in  which  this  ratio  has 
a  still  lower  ratio.  Besides,  however  lean  it  is,  meat  is  composed 
of  cells,  and  we  cannot  know  from  inspection  how  much  proteid 
it  contains,  nor  how  much  fat  or  glycogen  —  not  accurately  enough 
to  be  able  to  say  precisely  how  much  proteid  carbon  is  or  is 
not  retained  in  an  experiment.  Lean  meat  contains  both  fat 
and  glycogen  in  larger  quantities  than  the  methods  of  ex- 
traction in  use  at  the  time  of  Voit's  experiments  were  capable 
of  revealing.  In  2.5  kg.  of  lean  meat  it  is  not  likely  that 
there  was  less  than  10  g.  of  fat  ;  the  muscles  of  cats  and  rabbits 
after  very  careful  cleaning  contain  I  per  cent.  ;  and  according 
to  the  recent  analyses  of  Pfliiger  the  amount  of  glycogen  is  not 
likely  to  have  been  any  less.  It  is  clear,  therefore,  that  such 
experiments  do  not  furnish  a  secure  foundation  for  the  belief 
that  fats  are  made  from  proteids.  The  balance-sheet  method 
cannot  be  finely  enough  adjusted  to  decide  this  question. 

The  second  corner-stone  in  the  building  up  of  the  doctrine 
that  fat  could  be  derived  from  proteids  was  supplied  by  pathology. 
Virchow,  as  is  well  known,  distinguished  two  forms  of  fatty 
change  in  the  cells  of  the  principal  organs  of  the  body  :  a  fatty 
infiltration,  in  which  fat  imported  from  other  parts  was  deposited 
in  the  cells  ;  and  a  fatty  degeneration,  in  which  the  fat  was 
formed  in  the  cells  from  their  own  protoplasm.  The  need  for 
strict  proof  of  the  chemical  possibility  of  a  transformation  of 


S8  ASSIMILATION  AND  SYNTHESIS  OF  FAT        [LECT. 

proteid  into  fat  was  in  this  case  hardly  felt,  as  the  microscopical 
appearances  familiar  to  pathologists  seemed  to  point  so  clearly 
to  it.  Moreover,  the  same  change  was  believed  to  take  place 
when  milk  is  formed  in  the  mammary  gland,  or  sebum  in  the 
glands  of  the  skin,  when  the  brain  undergoes  softening,  or 
nerve  tissue  in  other  parts  degenerates,  and  when  living 
leucocytes  are  converted  into  dead  pus  corpuscles :  the  fats  of 
a  caseating  gland,  of  the  corpus  luteum,  or  of  an  encysted 
extra-uterine  gestation  were  all  supposed  to  have  a  similar 
origin,  and  were  derived  from  the  proteids  of  the  dying  cells. 

This  pathological  dogma  that  protoplasm  when  failing  in 
its  functions  tended  to  turn  into  fat,  though  so  generally 
accepted,  did  not  pass  unchallenged.  In  1883  Lebedeff  devised 
an  ingenious  experiment  to  determine  the  origin  of  the  fat  in 
the  liver  after  phosphorus  poisoning.  He  had  previously  shown 
that  dogs,  which  had  been  reduced  by  a  month's  starvation,  and 
after  that  fed  on  a  diet  containing  large  quantities  of  linseed 
oil  or  mutton  fat,  fattened  and  acquired  a  fat  that  in  the  former 
case  was  fluid  at  o°  C,  and  in  the  latter  was  still  solid  at  50°  C. ; 
in  both  cases  different  from  normal  dog's  fat,  but  similar  to 
the  fat  of  the  food.  If,  now,  such  a  dog  were  poisoned  with 
phosphorus  and  died  with  u  fatty  degeneration "  of  the  liver, 
upon  the  supposition  that  the  fat  of  the  diseased  liver  would 
be  formed  in  the  cells  of  the  liver  out  of  their  own  proteids,  it 
should  be  similar  to  that  in  the  liver  of  any  other  dog  that  had 
died  of  phosphorus  poisoning.  There  is  no  reason  why  fat 
made  from  the  proteids  of  the  liver  should  be  affected  by  the 
fact  that  an  abnormal  fat  happened  to  be  present  in  the 
subcutaneous  tissue.  But  if  the  fat  in  the  liver  were  imported 
from  other  parts  as  ready-made  fat,  then  it  should  be  similar 
to  the  fat  stored  in  these  parts.  In  a  dog  fattened  on  linseed 
oil  and  poisoned  with  phosphorus,  Lebedeff  showed  that  the 
fats  obtained  from  the  liver  consisted  largely  of  linolein.  He 
saponified  the  fat,  prepared  the  lead  salts,  separated  those 
soluble  in  ether  from  those  that  were  insoluble,  and  found  that 
the  former  gave  liquid  fatty  acids  corresponding  to  67  per  cent, 
the  latter  solid  acids  to  23  per  cent,  of  the  fat.  On  treating 


IV.] 


LEBEDEFF'S  EXPERIMENT 


89 


the  liquid  acids  with  nitrous  acid,  he  obtained  an  amount  of 
elaidic  acid  corresponding  to  only  one-fifth  of  these  acids.  The 
rest  he  argued  must  be  linoleic  acid,  which  does  not  solidify 
with  nitrous  acid.1 

Lebedeff  s  experiment  has  been  repeated  more  recently  by 
Rosenfeld,  and  also  by  Leick  and  Winkler  in  Krehl's  laboratory. 
The  estimation  of  linoleic  acid  in  liquid  fatty  acids  by  the 
elaidin  test  is  unreliable,  and  this  was  a  weak  point  in  Lebedeff  s 
argument  ;  for  even  olive  oil,  nearly  three-quarters  of  which 
consists  of  olein,  may  under  certain  conditions  yield  no  elaidin 
with  nitrous  acid.  In  the  later  modifications  of  this  experiment 
the  nature  of  the  fat  in  the  "degenerated"  organs  has  been 
determined  by  the  iodine  absorption.  Fats  containing  un- 
saturated  acids  like  oleic  or  linoleic  acid  absorb  iodine,  and  the 
amount  of  unsaturated  acid  can  be  determined  by  the  percentage 
amount  of  iodine  absorbed,  which  is  called  the  iodine  value  of 
the  fat.  Thus  Leick  and  Winkler  found  the  following  iodine 
values  for  fat  from  the  situations  indicated : 2 


Subcutaneous 
Fat. 

Myocardial 
Fat. 

56.1 

70.7 

Dog  poisoned  with  Phosphorus 

58.6 
38.2 

82.9 
58.2 

Sheep  poisoned  with  Phosphorus     .       •  .  i 

36.9 

64.I 

A  dog  was  then  starved  to  emaciation :  it  was  then  fed  on  a 
full  diet  of  meat  with  large  quantities  of  mutton  fat,  and  finally 
phosphorus  was  given.  The  subcutaneous  fat  after  death  had 
the  iodine  value  43.3,  and  the  fat  from  the  degenerated  heart 
muscle  64.6.  This  last  figure  is  almost  identical  with  that  found 
in  the  sheep  when  poisoned  with  phosphorus,  and  very  different 

1  Lebedeff,  Pfl.  A.  31,  11,  1883. 

2  Leick  and  Winkler,  S.  A.  48,  163,  1902. 


SO  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT. 

from  that  which  is  normal  for  the  dog  under  these  circumstances. 
Rosenfeld  obtained  similar  results  with  phlorrhizine,  which 
under  certain  conditions  also  causes  "  fatty  degeneration "  of 
the  liver.  He  pointed  out,  too,  that  the  liver  in  this  case,  though 
it  may  contain  from  25  to  even  75  per  cent,  of  fat,  does  not 
contain  more  than  a  gramme  or  two  less  proteid  than  it  should, 
and  that  the  accumulation  of  fat  can  very  rapidly  be  disposed 
of  and  the  disturbance  completely  cured.  This  could  hardly 
be  the  case  if  the  protoplasm  of  the  liver  cells  had  degenerated, 
and  this  great  mass  of  fat  was  formed  from  the  cell  proteids. 
Neither  is  it  likely  that  the  fat  is  derived  from  the  proteids  of 
other  organs ;  for  though  there  is  an  increased  output  of 
nitrogen,  and  therefore  proteids  are  broken  down  under  the 
action  of  the  drug,  the  carbon  excretion  is  also  increased,  owing 
to  the  severe  glycosuria,  and  there  is  no  retention  of  carbon.1 

A  further  important  point,  first  noted  by  Lebedeff  in  a 
case  of  phosphorus  poisoning  in  man,  has  been  confirmed 
experimentally  in  animals  by  Rosenfeld.  The  patient  whose 
case  is  referred  to  by  LebedefF  was  in  a  state  of  extreme 
emaciation  when  the  poison  took  effect,  and  at  the  post-mortem 
examination  he  observed  that  there  was  no  fatty  change  in  the 
liver.  Rosenfeld  showed  in  a  series  of  dogs  that  the  amount 
of  fatty  change  in  the  liver  after  phosphorus  poisoning  varied 
directly  with  the  amount  of  fat  in  the  body,  and  in  emaciated 
animals  there  might  be  none  appreciable.  A  similar  relation 
was  established  also  in  fowls. 

These  experiments  and  observations  prove  that  Lebedeff 
was  almost  unquestionably  right,  and  that  in  these  most  typical 
forms  of  fatty  degeneration  in  Virchow's  sense  the  fat  is  not 
produced  by  degradation  of  the  proteids  in  the  cells,  but 
imported  into  them  from  the  storage  places  of  the  body. 

Many  attempts  have  been  made  to  determine  this  point 
in  another  way,  by  ascertaining  whether  the  total  amount  of 
fat  in  the  body  is  increased  in  phosphorus  poisoning.  This 
can  only  be  done  by  comparing  poisoned  animals  with  normal 

1  Rosenfeld,  Z.f.  k.  M.  36,  232,  1898  ;  £/!,  too,  M.  /.,  p.  53,  1897  ;  and, 
Ergeb.  ii.,  p.  50  seq. 


iv.]  THE  FAT  IN  DISEASED  ORGANS  91 

animals,  a  method  which  can  only  give  uncertain  results.  In 
some  cases  the  answer  has  been  given  in  the  affirmative,  but 
not  in  the  most  convincing.  Athanasiu,  for  instance,  in 
Pfluger's  laboratory  took  124  pairs  of  frogs,  the  frogs  in  each 
pair  being  of  equal  size ;  one  frog  out  of  each  pair  was  given 
phosphorus,  the  other  serving  as  a  control.  No  appreciable 
difference  in  the  ratio  of  fat  to  body-weight  could  be  detected 
on  comparing  the  poisoned  frogs  with  the  normal  frogs.1  Kraus 
and  Sommer,  working  with  mice,  found  that  phosphorus  actually 
diminished  the  total  amount  of  fat  in  the  body,  although  by 
altering  its  distribution  the  amount  of  it  found  in  the  liver  was 
increased.2  This  observation  is  in  accord  with  the  account  of 
the  change  caused  by  phosphorus  which  was  given  by  Lebedeff 
and  by  Rosenfeld. 

It  remains,  therefore,  necessary  to  try  and  harmonise  this 
conclusion  with  all  the  other  instances  cited  by  Virchow  of  a 
similar  change,  by  which  proteids  were  believed  to  be  converted 
into  fats.  As  Rosenfeld  points  out,  Virchow  was  led  astray  by 
microscopic  appearances.  Virchow  speaks  of  the  brain  tissue, 
in  the  condition  known  as  yellow  softening,  entering  into  a 
state  exactly  similar  to  that  of  a  secreting  mammary  gland, 
drops  of  fat  being  secreted  in  the  protoplasm  in  each  case. 
"  When  milk  is  manufactured  in  the  brain  instead  of  in  the 
mamma,  the  process  is  a  form  of  softening  of  the  brain." 
Because  no  fat  drops  are  to  be  seen  in  normal  brain  tissue, 
it  does  not  follow  that  the  drops  when  they  appear  are  formed 
from  proteid.  But  Hoppe-Seyler3  and  Walther  proved  that 
degenerated  nerve  fibres,  though  densely  loaded  with  fat  drops, 
really  contain  less  fat  than  the  corresponding  normal  nerves 
from  the  other  side  of  the  body,  in  which  no  drops  of  fat  are 
visible  at  all.  Similarly,  Rosenfeld  compared  the  amount  of 
fat  in  a  patch  of  softening  in  the  occipital  lobe  on  one  side 
with  the  amount  in  the  corresponding  part  of  the  brain  on  the 
other  normal  side :  18  g.  of  softened  brain  tissue,  containing  3.06  g. 

1  Athanasiu,  Pfl.  A.  74,  511,  1899. 

2  Kraus  and  Sommer,  H.  B.  2,  86,  1902. 

3  Hoppe-Seyler,  V.  A.  8,  127  ;  Walther,  V.  A.  20,  426. 


92  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT, 

solid  matter,  gave  36.3  per  cent,  of  extract  (6.17  per  cent  of 
the  fresh  tissue);  16  g.  of  healthy  brain  substance  with  3.26  g. 
of  solids  containing  43.3  per  cent,  of  extract  (8.8  per  cent,  of 
the  fresh  tissue).  And  even  the  fat  in  the  secretion  of  the 
mammary  gland,  we  have  now  the  best  reasons  for  believing, 
is  not  transformed  proteid,  but  simply  fat  brought  to  the  gland 
by  the  blood  from  the  fat  stores  of  the  body.  For,  just  as  the 
fat  on  which  an  animal  has  been  fed  can  be  traced  into  the 
subcutaneous  tissue,  so  it  can  too  be  traced  into  the  milk. 
Cows  fed  on  maize  oilcake  give  butter  the  melting  point  of 
which  is  so  low  that  it  is  unsaleable.  If  animals  during  or 
before  lactation  are  fed  on  fats,  the  unsaturated  acids  of  which 
have  been  saturated  by  iodine,  these  same  fats  are  found  in 
the  milk,  retaining  their  iodine.1  Rosenfeld  fattened  a  bitch 
after  a  period  of  starvation  on  a  diet  rich  in  mutton  fat,  and 
then,  with  the  beginning  of  lactation,  fed  it  only  on  lean  meat. 
The  milk  contained  fat  the  iodine  value  of  which  was  practically 
that  of  mutton  fat.2  And  what  is  true  of  the  mammary  gland 
is  equally  true  of  the  sebaceous  glands.  The  secretion  of  these 
glands,  which  Virchow  regarded  as  the  product  of  a  fatty 
degeneration  of  protoplasm,  has  been  proved  by  Plato  and  by 
Rohmann  to  contain  the  fats  which  are  present  in  the  food. 
Sesame  oil  gives  a  characteristic  colour  reaction  with  hydro- 
chloric acid  and  furfurol.  In  geese  that  have  been  fed  on  this 
oil,  the  secretion  of  the  sebaceous  glands  about  the  tail  also 
gives  this  reaction,  and  when  tested  by  determinations  of  iodine 
value,  melting  point,  and  molecular  weight,  the  nature  of  the 
fatty  acids  present  in  the  secretion  is  found  to  vary  according 
to  the  fat  in  the  food.3 

But  it  is  not  only  in  nerve  tissue  and  the  glands  which 
secrete  fat  that  microscopic  appearances  have  led  to  miscon- 
ceptions. Normal  human  cardiac  muscle  contains  no  fat  that 
is  visible  under  the  microscope ;  but  according  to  Rosenfeld's 
analyses  15  per  cent  of  the  solid  matter  in  it  is  fat  The 

1  Winternitz,  H.-S.  Z.  24,  425,  1898. 

2  Rosenfeld,  Allgem.  Med.  Central.  Zeitung,  No.  60,  1897. 

3  Rohmann,  H.  B.,  v.,  no,  1904. 


iv.l         FATTY  COMPOUNDS  NOT  STAINING  AS  FAT 


93 


kidney — in  which  fat  cannot,  either,  be  detected  microscopically 

contains  still  more  than  the  heart,  about  18  per  cent,  by  the 
same  method  ;  and  a  fatty  kidney  contains  no  more  than  a 
normal  one.  Dudgeon  has  made  preparations  of  the  hearts  of 
guinea-pigs  that  were  killed  by  injection  of  diphtheria  toxin. 
Unlike  the  normal  heart  muscle,  these  preparations  show  very 
large  quantities  of  fat  when  stained  with  scharlach.  The 
difference  is  most  striking,  and  suggests  that  the  tissue  must 
contain  a  very  great  deal  more  fat  than  the  normal  muscle, 
which,  in  fact,  shows  with  scharlach  no  sign  of  containing  fat 
at  all.  Dr  Dudgeon,  however,  allowed  me  to  determine  the 
amount  of  fat  in  some  of  these  hearts.  Twelve  guinea-pigs 
were  taken,  six  of  which  received  fatal  doses  of  diphtheria 
toxin  and  died  within  forty-eight  hours.  The  other  six,  of 
approximately  the  same  size,  were  then  killed.  The  hearts  of 
the  guinea-pigs  that  had  been  killed  by  the  toxin  stained 
intensely  with  scharlach,  the  others  not  at  all.  The  results  of 
the  analysis  were  as  follows  : — 


Extract 

Weight  of 
Fresh- 
muscle 
Substance. 

Weight, 
Dry  and 
Powdered. 

Ether   ^ 
Extract,  | 
Rosen-    >- 
feld's 
method,  j 

f     Per 

1  cent,  of 
=  •{     Dry 
Sub- 
\.  stance. 

Saponified. 
Insoluble  "\ 
Fatty 
Acids      V 
from 

{Per 
cent,  of 
Dry 
Sub- 

Extract. ) 

stance. 

Hearts   of  Six 

normal  Guinea- 

pigs 

6.542 

1.4061 

0.2451 

1743 

0.1344 

9-55 

Six  poisoned 

Guinea-pigs     . 

10.945 

1.9997 

0-3954 

19.77 

0.2440 

12.20 

These  figures  show  that  the  hearts  in  which  the  toxin  had 
set  up  changes  contained  more  fats  than  the  others ;  according 
to  the  last  column,  about  25  per  cent.  more.  But  the  dried 
normal  hearts  contained  17.43  Per  cent-  J  anc*  yet  no  trace  of 
this  could  be  detected  microscopically.  In  this  case  it  is  clear 
that  the  toxin  causes,  in  addition  to  an  accumulation  of  fat 
which  is  not  very  great,  changes  in  the  condition  in  which  the 


94  ASSIMILATION  AND  SYNTHESIS  OF  FAT         [LECT. 

fat  is  present  in  the  muscle  substance.  That  the  accumulation 
of  fat  was  not  sufficient  to  account  for  the  microscopical  appear- 
ances is  practically  certain — reckoned  on  the  fresh  tissue,  the 
increase  in  higher  fatty  acids  was  from  2.05  per  cent,  in  the 
normal  hearts  to  2.23  per  cent,  in  those  on  which  the  toxin  had 
acted.  It  is  difficult  to  believe  that  the  intense  scarlet  staining 
of  the  latter  was  due  to  a  substance  that  formed  only  about 
0.2  per  cent,  of  the  tissue.  The  probability  is,  that  as  in  the 
fatty  kidney,  in  which  Rosenfeld  could  detect  no  abnormal 
amount  of  fat,  and  as  in  the  degenerated  nerve  tissue,  the 
manner  in  which  the  fatty  acids  are  combined  is  altered  by  the 
poison.  In  the  normal  heart,  kidney,  or  nerve,  the  fatty  acids 
are  not  present  as  simple  glycerides :  they  are  combined  in 
such  a  way  that  they  do  not  react  histologically  as  fat.  Under 
the  influence  of  diphtheria  toxin  these  combined  fats  or  fatty 
acids  are  set  free  from  the  heart  substance  as  they  are  in 
degenerating  nerves,  and  then  they  answer  to  the  histological 
tests  for  fat.  If  we  extend  the  term  protoplasm  to  include 
these  unknown  compounds  of  fat,  such  as  the  myelin  of  nerve 
tissue,  and  if  we  do  not  regard  protoplasm  as  a  synonym  for 
proteids,  then  we  may  retain  the  term  fatty  degeneration  of 
protoplasm  for  the  pathological  changes  described  by  Virchow : 
only  it  must  always  be  quite  clear  that  the  fat  is  fat  that  has 
been  unmasked,  when  it  is  not  fat  that  has  been  imported,  and 
that  it  certainly  is  not  fat  that  has  been  made  in  the  cells  by  a 
chemical  change  from  ordinary  proteid  substances. 

Bainbridge  has  described  the  same  histological  change  in 
the  liver  after  ligature  of  the  hepatic  artery.  The  normal  liver 
tissue  of  cats  stains  very  slightly  with  scharlach.  But  after 
ligature  of  the  artery  within  forty-eight  hours  there  are  marked 
changes  in  the  microscopic  appearance  of  the  tissue  when 
treated  with  this  reagent,  the  peripheral  parts  of  the  lobule 
particularly  staining  intensely.  The  analyses,  however,  show 
that  these  abnormal  livers  frequently  contain  less  fat  than  the 
normal  ones  which  hardly  give  any  histological  reactions 
for  fat. 

The  evidence,  therefore,  for  the  formation  of  fat  from   the 


iv.]  THE  SOURCE  AND  ORIGIN  OF  GLYCERINE  95 

proteids  of  the  body  has  melted  away,  no  less  than  that  for  the 
transformation  of  the  proteids  in  food  into  fat.  But  to  admit 
this,  is  not  the  same  as  to  say  that  such  a  transformation  of 
material  cannot  occur.  The  removal  of  nitrogen  from  proteid 
substances,  which  was  hypothesised  by  the  school  of  Voit  in 
their  account  of  fat  formation,  has  become  an  established  fact 
in  metabolism,  owing  to  the  work  of  Lang  and  others ;  and 
though  we  cannot  say  that  the  carbon  compounds  left  after  the 
amido  acids  have  lost  their  nitrogen  are  retained  and  converted 
into  fat,  we  cannot  say  that  this  can  never  be  the  case.  If 
alanine  were  set  free  from  all  the  compounds  in  which  it  occurs 
in  proteids  and  by  the  loss  of  its  NH2  group  were  converted  in 
lactic  acid,  this  is  the  same  substance  that  we  have  felt  compelled 
to  regard  provisionally  as  the  connecting  link  between  carbo- 
hydrates and  fats.  And  at  any  rate  we  do  not  know  enough 
about  the  subsequent  fate  of  denitrified  products  of  proteid 
hydrolysis  to  justify  a  direct  negation  of  the  possibility  of  the 
formation  of  fat  from  some  of  them,  or  from  substances  derived 
from  some  of  them.  All  we  can  say  is,  that  what  has  sometimes 
passed  as  proof  of  the  change  has  not  stood  the  tests  to  which 
it  has  been  submitted. 

Lastly,  with  regard  to  the  origin  of  glycerine,  there  can  be 
no  doubt  that  this  is  a  substance  that  can  be  formed  in  the 
ordinary  course  of  metabolism.  Munk's  experiments  on  the 
absorption  of  free  fatty  acids  and  their  appearance  as  glycerides 
in  the  chyle  show  this.  The  fact,  too,  that  fats  are  synthesised 
from  carbohydrates  implies  that  the  glycerine  as  well  as  the 
acids  are  so  synthesised,  since  glycerine  is  not  commonly  intro- 
duced into  the  body  except  in  the  form  of  neutral  fats.  The 
experiments  of  Liithje l  have  made  it  practically  certain  that 
glycerine  can  be  converted  into  sugar  in  the  body,  and  there  is, 
therefore,  good  ground  for  regarding  sugar  as  a  source  from 
which  the  glycerine  in  the  body,  when  not  derived  from  the  fats 
of  the  food,  takes  its  origin.  If  glyceric  aldehyde  is  the  first 
derivative  from  sugar  in  the  production  of  lactic  acid,  the  change 
from  the  aldehyde  to  the  alcohol  is  one  that  is  not  improbable. 
1  Liithje,  D.  A.f.  k.  M.  80,  101,  1904  ;  cf.  supra,  p.  46. 


96  ASSIMILATION  AND  SYNTHESIS  OF  FAT     [LECT.  iv. 

J.  Schmid,  in  three  experiments  on  dogs  treated  with  phlorrhizine, 
found  that  fatty  acids  added  to  a  meat  diet  diminished  the 
excretion  of  sugar.1  Since  the  animals  had  been  treated  with 
phlorrhizine  for  some  days  they  probably  contained  but  little 
glycogen.  The  fatty  acids,  therefore,  in  these  cases,  combined 
with  glycerine  which  was  derived  from  material  which  was  not 
carbohydrate,  but  which  would  otherwise  have  been  converted 
into  sugar.  No  other  experimental  evidence  on  this  point  is  at 
present  available. 

1  J.  Schmid,  5.  A.  53,  429,  1905. 


LECTURE  V 

THE   CATABOLISM   OF   FAT 

THE  assimilation  and  physiological  synthesis  of  fat  have  long 
occupied  much  of  the  attention  of  physiologists,  as  the  familiar 
discussions  on  the  formation  of  fat  from  carbohydrates  and 
from  proteids,  and  on  the  problems  of  obesity  and  pathological 
fatty  change  bear  witness.  The  subsequent  fate  of  fat,  whether 
assimilated  or  made  in  the  body,  presented  apparently  fewer 
points  of  interest.  It  was  sometimes  taught  that  fat  was 
principally  of  value  as  a  source  of  heat,  and  that  in  the  arctic 
regions  a  lining  of  fat  was  more  effectual  than  furs  and  blankets 
in  preventing  the  loss  of  body-heat.  But  in  what  organs  the 
evolution  of  heat  from  fat  took  place,  where  the  long  fatty  acid 
chains  were  taken  to  pieces  and  oxidised ;  how  such  changes 
were  brought  about,  and  what  functions,  if  any,  were  served 
by  them  in  addition  to  that  of  supplying  the  body  with  heat 
— these  were  questions  on  which  curiosity  received  but  little 
encouragement. 

But  it  is  a  fact  that  is  now  generally  appreciated,  that  the 
fat  molecules  are  at  least  as  important  physiologically  as  the 
carbohydrate.  In  the  experiments  carried  out  on  the  pro- 
fessional starving  man,  Cetti,  it  was  found  that  nitrogenous 
metabolism  calculated  per  unit  of  body-weight  remained  fairly 
constant  between  the  fifth  and  tenth  days  of  starvation,  ranging 
between  0.15  and  0.20  g.  daily:1  on  an  average,  therefore,  a 
little  over  I  g.  of  proteid  per  kg.  daily.  To  convert  this  amount 

1  Zuntz  and  Lehmann,  B.  k.  W.t  No.  24,  1887. 
97  G 


98  THE  CATABOLISM  OF  FAT  [LECT. 

of  proteid  into  urea,  carbonic  acid,  sulphuric  acid,  and  water, 
nearly  2  g.  of  oxygen  would  be  required,  having  the  volume 

22 

-^-=1.4  litre,  which  amount,  in  twenty-four  hours,  is  at  the  rate 

of  nearly  I  c.c.  per  minute.  But  Cetti's  oxygen-consumption 
was  at  the  rate  of  nearly  5  c.c.  per  kg.  minute  ;  so  that  four- 
fifths  of  the  oxygen  absorbed  was  required  for  the  oxidation  of 
non-nitrogenous  substances.  A  gramme  of  oxygen  yields  very 
nearly  the  same  number  of  calories  whether  it  is  used  for  the 
oxidation  of  proteid,  carbohydrate  or  fat ;  so  that  four-fifths  of 
the  starving  man's  energy  was  derived  from  non-nitrogenous 
material,  much  the  greater  part  of  which  must  have  been  fat 
during  the  greater  part  of  the  time  in  which  these  observations 
were  made.  Cetti  was  not  well  stocked  with  fat  when  he  began 
this  fast,  and  his  nitrogen  excretion  was  higher  than  that 
recorded  in  other  cases  of  inanition.  In  dogs,  Rubner  and  Voit 
reckon  that  during  starvation  only  from  10  to  16  per  cent,  of 
the  total  energy  is  derived  from  proteids,  the  rest,  up  to  90  per 
cent,  that  is,  being  derived  from  fat. 

It  is  clear,  therefore,  that,  at  any  rate  in  starvation,  arrange- 
ments exist  by  which  the  greater  part  of  the  energy  needed  for 
life  can  be  obtained  from  the  oxidation  of  fats.  How  much  of 
the  total  chemical  energy  transformed  in  metabolism  is  con- 
verted directly  and  in  the  first  instance  into  heat,  and  how  much 
primarily,  if  not  finally,  into  other  forms  of  energy,  is  not  known  ; 
but  it  is  difficult  to  suppose  that  none  of  the  work  done  by  the 
heart,  the  respiratory  muscles,  kidneys,  or  other  glands  is 
performed  by  means  of  the  oxidation  of  fat.  Zuntz  reckons 
that  the  functions  of  the  circulation  and  respiration  by  them- 
selves account  for  from  10  to  20  per  cent,  of  the  total  energy 
expenditure  in  starvation.1  It  is  remarkable,  too,  that  the 
organs  which  have  the  most  unceasingly  continuous  work  to  do 

1  Cit.  v.  Noorden,  Pathologische  Stoffwechsel^  p.  97.  The  work  done  by 
the  heart  is  reckoned  to  be  equal  to  about  27,000  kg.  m.  Supposing  the 
efficiency  of  heart-muscle  as  a  working  machine  to  be  33  per  cent,  then 
27,000  kg.  m.  work  x  3  =  200  Cal.  =  nearly  50  g.  proteid— nearly  the  whole 
proteid  metabolism  of  the  body  in  Cetti. 


v.]  THE  USE  OF  FAT  IN  THE  BODY  99 

are  richly  stocked  with  fat.  In  the  human  heart  muscle,  15  per 
cent,  of  the  solids  are  soluble  in  ether,  and  more  than  half  of 
the  ether  extract  is  composed  of  fat :  this  would  be  enough  by 
itself  to  supply  the  heart  with  fuel  for  six  or  seven  hours'  work. 
In  the  kidney,  according  to  Rosenfeld,  there  is  still  more  fat 
than  in  the  heart  Of  the  voluntary  muscles  the  diaphragm, 
and  in  the  rabbit  the  deeply  placed  red  muscles,  such  as  the 
soleus,  pectineus,  crureus  and  semi-tendinosus,  which  are 
believed  to  be  especially  concerned  with  the  more  protracted 
forms  of  muscular  activity,  differ  from  the  paler,  more  super- 
ficial muscles  in  containing  considerably  more  fat,1  though  less 
than  the  heart.  Attempts  to  demonstrate  a  diminution  in  the 
amount  of  fat  in  the  muscles  during  activity  have  been  made ; 
both  sciatic  nerves  have  been  divided,  and  one  of  them  tetanised 
for  some  hours,  and  the  fat  in  the  corresponding  muscles  on  the 
two  sides  compared.  But  no  evidence  for  the  immediate  con- 
sumption of  fat  in  muscular  activity  has  been  obtained  in  this 
way.  In  one  experiment  of  this  kind,  I  found  that  both  in  the 
gastrocnemius  and  the  tibiales  on  the  tetanised  side,  there 
was  rather  more  fat,  reckoned  in  percentage  of  the  dry  sub- 
stance, than  on  the  side  that  had  been  kept  at  rest. 
Zuntz  and  Bogdanoff  found  that  the  effect  of  stimulating 
muscles  was  to  diminish  in  them  the  amount  of  those  com- 
binations of  fatty  acids  which  cannot  be  extracted  directly  with 
ether.2 

It  is  doubtful  whether  the  utilisation  of  fat  in  muscular 
activity  can  be  proved  by  stimulating  the  muscles  with  the  circu- 
lation normally  maintained  through  them.  But  in  other  ways 
it  has  been  established  beyond  doubt  that  the  muscles  can  and 
do  make  use  of  fat  as  a  source  of  energy.  To  begin  with,  Zuntz 
and  his  pupils  have  shown  that  muscular  activity  does  not  alter 
the  respiratory  quotient  unless  the  work  is  severe  enough  to 
interfere  with  the  oxygen-supply  to  the  muscles.  This  may 
hold  when  the  work  done  is  sufficient  to  increase  the  oxygen 
consumption  more  than  threefold.  This  great  increase  in  the 

1  Leathes,  //.  of  Phys.,  xxxi.,  p.  ii.,  1904. 

2  Zuntz  and  Bogdanoff,  D.  R.  A.  13,  1897  ;  Pfl.  A.  65,  8 1. 


100 


THE  CATABOLISM  OF  FAT 


LECT 


rate  of  oxidation  in  the  body  is  unaccompanied  by  any  increase, 
or  at  least  any  material  increase,  in  the  nitrogen  output :  so 
that  the  energy  must  be  supplied  by  non-nitrogenous  material. 
If  this  increased  metabolism  involved  only  carbohydrates,  the 
respiratory  quotient  must  be  raised :  since  it  is  not,  fat  as  well 
as  carbohydrate  must  be  made  use  of  to  supply  the  muscles 
with  what  they  require  for  their  work. 

Even  in  fasting  animals  there  is  only  very  little  increase  in 
the  excretion  of  nitrogen  when  they  are  made  to  work.  A  dog 
on  the  sixth  and  seventh  days  of  starvation  was  made  to  do 
work  in  a  treadmill  equivalent  to  climbing  to  the  height  of  1400 
metres.  The  nitrogen  excretion  rose  from  6  g.  to  6.6.  The 
energy  for  this  work  must  have  been  derived  in  the  main 
from  fat. 

Zuntz,  in  fact,  finds  that  fat  can  be  used  for  muscular  work  no 
less  economically  than  either  proteids  or  carbohydrates :  he 
determined  the  oxygen-consumption  and  respiratory  quotient 
in  a  man  resting  and  working  on  three  different  diets — one 
principally  fat,  one  principally  carbohydrate,  and  the  other 
principally  proteid — and  found,  as  the  results  in  the  following 
table  show,  that  slightly  less  oxygen  and  energy  was  required  to 
do  work  on  the  fat  diet  than  on  the  others. 


Diet 

principally. 

Res' 

c.c. 
Oxygen 
used 
per  min. 

'ing- 

Working. 

M.  Kg. 
of  Work 
done. 

PerM.  Kg.  of  Work. 

Resp. 
Quotient. 

c.c. 

Oxygen 
used 
per  min. 

Resp. 
Quotient. 

c.c. 
Oxygen 
used. 

Cal. 

Fat  . 

319 

0.72 

I02Q 

0.72 

354 

2.01 

9-39 

Carbohydrate  . 

277 

0.9 

1029 

0.9 

346 

2.17 

10.41 

Proteid     . 

306 

0.8 

1127 

0.8 

345 

2.38 

11-35 

Similarly,  in  dogs,  even  after  a  period  of  starvation  followed 
by  phlorrhizine  glycosuria,  when  the  carbohydrate  stores  must 
have  run  very  low,  work  is  done  on  the  fat  of  the  body  as 


v.]  WORK  DONE  IN  THE  MUSCLES  WITH  FAT          101 

economically    as    when    proteid    food    or    carbohydrates    are 
abundant.     In  the  following  table  this  is  shown. 


Diet,  etc. 

Resp. 

Quotient. 

Oxygen 
used 
per  M.  Kg. 

Heat-equivalent 
of  this  amount 
of  Oxygen. 

i.  Proteid  diet  »  , 

O.78 

O  C7  r  C 

2  C8    f-il 

2.  Proteid  diet,  but  no  food  given  on  day 
of  experiment     

0.72 

0.53  „ 

2-43      „ 

3.  As  in  2,  only  Sugar  was  given  before 

O  ^4. 

2  C8 

4.  Little  Proteid,  much  Starch  . 

0.88 

°.55    „ 

*'$0          ,, 

2.6          „ 

5.  Starved  :  Phlorrhizine  glycosuria 

0.71 

0-59   «, 

2.71    „ 

In  the  fifth  of  these  experiments  certainly  the  work  must 
have  been  done  with  energy  derived  from  fat,  and  from  the  con- 
sumption of  oxygen  and  the  calculated  expenditure  of  energy 
it  is  clear  that  it  was  done  as  economically  as  in  the  other 
experiments. 

The  fats,  then,  can  be  used  as  a  source  of  energy  by  muscles 
as  well  as  proteids  and  carbohydrates,  and  the  yield  of  work  for 
a  given  amount  of  chemical  energy  in  the  form  of  fat  is  as  good 
as  in  the  case  of  either  of  the  other  kinds  of  material.1 

Of  the  physiological  importance  of  the  catabolic  changes  in 

1  It  has  been  shown  that  the  carbonic  acid  output  of  fowls'  eggs  from 
the  ninth  day  of  incubation  on  is  weight  for  weight  equal  to  that  of  the 
fully  grown  bird,  that  the  respiratory  quotient  is  about  0.7,  and  that  of 
the  5  or  6  g.  of  ether  extract  that  can  be  obtained  from  a  fresh  egg  about 
one-half  is  used  up  before  the  chick  is  hatched.  The  heat-loss  from  the 
egg  in  an  incubator  must  be  considerably  less  than  in  the  bird  at  large, 
although  evaporation  causes  some  loss  of  heat  that  must  be  replaced.  The 
oxidation  of  fat,  therefore,  must  be  needed  for  other  transformations  of  energy 
than  heat -production  ;  the  work  done  in  the  contractions  of  the  heart  is  all 
converted  back  into  heat,  so  that  it  is  difficult  with  the  data  which  are 
available  to  account  for  the  loss  of  chemical  energy  during  incubation.  Bohr 
and  Hasselbalch,  M.  J.,  p.  522,  1899  ;  Liebermann,  M.  /.,  p.  234,  1888. 


102  THE  CATABOLISM  OF  FAT  [LECT. 

fat  there  can  be  no  question,  whatever  the  use  made  of  these 
changes  in  the  economy  of  the  organism  may  be.  But  when  we 
attempt  to  form  a  conception  of  the  way  in  which  these  changes 
are  brought  about,  we  are  confronted  with  the  greatest  difficulties. 
The  hydrolysis  of  fats,  resulting  in  the  separation  of  the 
glycerine  from  the  fatty  acids,  it  is  true  we  know  something 
about :  but  this  hardly  touches  the  fringe  of  the  subject.  Until 
we  know  how  the  fatty  acids  are  oxidised,  we  do  not  know  the 
essential  part  of  the  process.  Our  ignorance  is  fundamental. 
And  it  stretches  into  all  departments  of  metabolism.  If  we  do 
not  know  how  simple  carbon  chains  are  oxidised,  we  can  form 
no  conception  of  the  way  in  which  those  transformations  of 
chemical  energy  which  constitute  life  are  brought  about  in  the 
metabolism  of  carbohydrates  or  proteids  any  more  than  in  that 
of  the  fats  themselves.  In  the  case  of  the  carbohydrates,  we 
have  already  seen  that  it  is  at  this  point  at  any  rate  that  we  are 
brought  up  to  a  stop.  For  even  supposing  that  it  is  right  to 
fancy  that  we  can  trace  sugar  metabolism  through  lactic  acid  to 
aldehyde  and  formic  acid,  the  energy  of  these  products  is  the 
same  as  that  of  the  sugar  from  which  they  were  derived ;  and ' 
assuming  that  the  aldehyde  becomes  acetic  acid,  this  would  still 
leave  540  calories  out  of  the  original  677,  or  about  80  per  cent 
of  the  energy  of  the  sugar,  still  latent  in  the  acetic  and  formic 
acids ;  and  the  whole  kernel  of  carbohydrate  metabolism  would 
lie  in  the  oxidation  of  the  simple  fatty  acids.  And  the  same 
holds  for  the  metabolism  of  proteids,  in  so  far  as  that  metabolism 
is  concerned  in  the  evolution  of  energy  from  chemical  combina- 
tions. For  there  is  good  ground  for  thinking  that  the  cleavage 
products  of  proteids  to  a  great  extent  lose  their  nitrogen  com- 
paratively early  in  the  course  of  their  catabolism,  and  it  is  a  fact 
that  the  removal  of  the  nitrogen  hardly  affects  the  energy  of  the 
molecules  ;  consequently  the  crucial  stage  in  proteid  catabolism 
consists  in  the  oxidation  of  simple  fatty  acids.  Physiological 
chemistry  teaches  us  to  look  upon  life  as  the  transformation  of 
chemical  energy  into  energy  of  a  different  nature,  in  conformity  to 
the  requirements  of  the  organism.  But  it  fails  us  just  where  its 
work  would  begin  to  have  significance  unless  it  can  help  us  to 


v.]  THE  OXIDATION  OF  FATTY  ACIDS  103 

follow  the  processes  by  which  the  simple  fatty  acids  are  made 
to  render  up  their  great  stores  of  energy. 

In  the  oxidation  of  fatty  acids  there  are  reasons  for  thinking 
that  the  problems  are  essentially  the  same  whether  we  have 
stearic  or  butyric  acid  to  deal  with ;  that  is  to  say,  that  the  long 
chains  are  step  by  step  reduced  in  length  by  a  process  which 
is  simply  repeated  at  each  step.  It  is  at  any  rate  true  that 
the  intermediate  members  of  the  fatty  acid  series  present 
chemical  combinations  in  which  the  energy  is  just  as  available 
for  the  purposes  of  the  body  as  those  commonly  taken  in  fatty 
food  Palmitin  is  no  less  valuable  as  a  food  than  stearin,  and 
in  Salkowski's  laboratory  Ludwig  Meyer  proved  that  lauric 
acid,  C12H24O2,  and  myristic  acid,  C14H28O2,  keep  down  proteid 
metabolism  just  like  the  higher  members  of  the  fatty  acid  series, 
and,  allowing  for  their  lower  potential  energy  and  indifferent 
absorption,  are  effective  foodstuffs.1  From  stearic  acid,  therefore, 
down  to  lauric  acid  the  fatty  acids  are  interchangeable.  Of 
the  lower  fatty  acids,  it  is  known  that  butyric,  valerian ic,  and 
caproic  acids,  when  given  with  food,  are  not  excreted  in  the 
urine;  whereas,  of  the  acetic  acid  contained  in  25  g.  of  sodium 
acetate  given  by  the  mouth,  about  10  per  cent,  and  of  the  formic 
acid  in  25  g.  of  sodium  formate,  about  26  per  cent,  can  be 
recovered  from  the  urine.2  When  caproic  acid  is  injected  into 
the  veins  it  is  oxidised  ;  acetic  and  formic  acid  under  these  con- 
ditions are  not3  It  is  also  known  that,  while  an  economy  of 
proteid  effected  by  lactic  acid  can  be  detected,  this  is  not  the 
case  with  acetic  acid  :  acetic  acid  is  not  completely  oxidised,  and, 
acting  as  a  diuretic,  actually  increases  the  amount  of  nitrogen 
excreted.4  But  if  this  acid,  when  absorbed  into  the  blood  from 
the  intestine,  or  injected  directly  into  a  vein,  is  not  all  of  it  avail- 
able as  a  source  of  energy  in  the  body,  it  does  not  follow  that 
when  generated  in  the  organs  in  the  course  of  their  metabolism 
it  is  not  capable  under  these  conditions  of  supplying  the  cells 

1  L.  Meyer,  H.-S.  Z.  40,  550,  1904. 

2  Schotten,  H.-S.  Z.  7,  375,  1883. 

3  Scheremetjewski,  B.  der  Sachs.  Ges.  der  Wiss.,  p.  154,  1868. 

4  Weiske  and  Flechsig,  M.  /.,  p.  408,  1889. 


104  THE  CATABOLISM  OF  FAT  [LECT. 

with  all  its  energy.  In  so  far  at  any  rate  as  the  acids  from 
stearic  to  lauric  are  all  equally  available  as  foods,  it  is  fair  to 
assume  that  the  reactions  by  which  stearic  acid  is  made  to 
yield  up  its  energy  are  equally  applicable  to  palmitic,  myristic, 
and  lauric  acids.  The  fact  that  it  is  only  the  even  number 
members  of  the  series  of  fatty  acids  above  caproic  acid  that 
occur  in  Nature,  and  the  fact  that  all  of  these  are  found  in 
milk,  may  possibly  indicate  that  the  demolition  of  the  higher 
fatty  acids  is  effected  by  the  removal  of  two  carbon  atoms  at 
a  time;  just  as  when  oleic  acid  is  fused  with  potash,  acetic 
acid  is  split  off  and  palmitic  acid  formed. 

Otherwise,  we  may  bear  in  mind  that  Le  Sueur  has  shown 
that  the  fatty  acids  may  be  derived  in  succession  from  each 
other  by  changes  in  which  the  chain  of  carbon  atoms  are 
shortened  by  one  atom  at  a  time.  The  a-oxy-fatty  acids,  oxy- 
stearic  no  less  than  lactic  acid,  when  heated  to  260  to  280°  C. 
split  up  into  the  next  lower  aldehyde  and  formic  acid,  or 
carbon  monoxide  and  water.  By  the  oxidation  of  the  aldehyde 
the  acid  is  obtained,  which  can  then  be  converted  into  the 
a-oxy-acid  and  the  process  repeated.  The  reaction  is  the  same 
at  each  step,  and  can  begin  with  lauric  acid  or  any  other  acid 
in  the  series  equally  well  as  with  stearic  acid.  In  that  case, 
oleic  acid  may  figure  as  the  first  derivative  from  stearic  acid, 
and  by  taking  up  water  give  rise  to  the  oxy-acid.  Only  it 
must  be  borne  in  mind  that  the  unsaturated  linkage  in  oleic 
acid  is  not  in  the  a-/3  position,  as  was  formerly  supposed,  from 
the  fact  that  on  fusion  with  potash  it  yields  palmitic  and  acetic 
acids,  and  not  in  the  /3-y  position,  as  has  also  been  thought, 
but  is  known  to  be  exactly  in  the  middle — 

C8Hir .  CH  :  CH .  C7H14 .  COOH. 

For  by  more  than  one  reaction  it  can  be  made  to  yield  on 
oxidation  normal  nonylic  acid  (pelargonic  acid)  and  azelaic 
acid,  COOH  .  C7H14.  COOH.1  But  that  the  unsaturated  linkage 
can  be  transferred  under  certain  conditions  from  the  centre  of 

1  Baruch,  B.  27,  173,  1894  ;  and,  Le  Sueur,  /.  C.  S.}  p.  1708,  1904. 


v.]  OXIDATION  OF  THE  /3-CARBON  ATOM  105 

the  molecule  to  the  «-/3  position,  seems  to  be  suggested  by  the 
result  of  potash  fusion. 

The  balance  between  these  two  alternatives — the  removal 
of  two  carbon  atoms  or  of  one  at  each  step — may  perhaps  be 
affected  by  some  very  interesting  results  recently  published 
by  Knoop  on  the  fate  of  aromatic  derivatives  of  the  lower 
fatty  acids  in  the  body.1  When  phenyl-acetic  acid  is  given  to 
a  dog,  it  combines  like  benzoic  acid  with  glycocoll,  and  is 
excreted  as  phenaceturic  acid — 

/H 
CH9 .  CO .  N< 

\CH2 .  COOH 


the  side  chain  is  not  oxidised.  Phenyl-propionic  acid  is 
oxidised  in  the  side  chain,  two  carbon  atoms  are  removed,  and 
it  is  excreted  as  hippuric  acid.  Phenyl-butyric  acid  is  also 
oxidised  in  the  side  chain,  but  appears  in  the  urine,  not  as 
hippuric  but  as  phenaceturic  acid ;  here  too,  therefore,  two 
carbon  atoms  are  removed,  but  not  the  third.  Phenyl-valerianic 
acid,  however,  gives  hippuric  acid  ;  four  carbon  atoms  are  in 
this  case  struck  off.  In  each  instance,  therefore,  the  oxidation 
appears  to  be  effected  at  the  /3-carbon  atom ;  in  the  case  of 
phenyl-valerianic  acid  this  occurs  twice,  and  when  there  is  no 
/3-carbon  atom  no  oxidation  takes  place.  We  cannot,  of  course, 
say  that  the  oxidation  of  these  aromatic  derivatives  of  the 
fatty  acids  is  necessarily  worked  out  on  the  same  lines  as  that 
of  the  fatty  acids  themselves.  But  the  influence  of  the  aromatic 
group  must  be  diminished  the  longer  the  side  chain  is,  and  the 
fate  of  phenyl-valerianic  acid  is  certainly  very  suggestive. 

If  we  may  argue  from  these  most  interesting  results,  then 
the  oxidation  of  stearic  acid  must  lead  to  palmitic,  of  palmitic 
acid  to  myristic,  and  so  on  down  the  series ;  and  the  reason 
why  we  can  find  traces  of  each  of  the  even  number  members 
in  this  series,  but  none  of  the  others,  is  at  once  apparent. 

The  higher  fatty  acids  are  extraordinarily  stable  compounds. 

1  Knoop,  H.  B.,  vi.,  150,  1905. 


106  THE  CATABOLISM  OF  FAT  [LECT. 

Tt  is  said  that  palmitic  and  stearic  acids  have  been  obtained 
from  tombs  in  Egypt,  where  they  must  have  been  for  several 
thousand  years.  Micro-organisms  do  not  live  in  pure  fats  or 
oils,  though  funguses,  especially  Penicillium  glaucuin,  grow  and 
thrive  on  moist  material  which  is  sunk  in  oil.  Apparently  they 
make  use  of  the  oil  to  some  extent  as  food,  hydrolysing  it, 
probably  removing  glycerine,  and  leaving  some  at  any  rate 
of  the  free  fatty  acid  to  crystallise  out.1  The  changes  that 
fats  and  oils  undergo  when  they  become  rancid  are  not  the  work 
of  micro-organisms.  Dry,  sterilised  fats  become  rancid  on 
exposure  to  air  and  sunlight.2  These  changes,  which  are  more 
prone  to  occur  the  more  unsaturated  acids  such  as  oleic  acid 
are  present  in  the  fat,  consist  partly  in  the  formation  of  the 
volatile  lower  fatty  acids,  and  partly  in  other  changes  the  nature 
of  which  is  not  understood.  Those  fats  or  oils  that  contain 
the  doubly  or  trebly  unsaturated  acids  linoleic  and  linolenic  acid, 
undergo  changes  which  are  referred  to  as  "  drying,"  and  which 
result  in  the  formation  of  a  varnish.  In  this  case  there  is  a  gain 
in  weight  from  absorption  of  oxygen.  But  the  products  of  the 
change  and  the  nature  of  the  reactions  by  which  they  are  pro- 
duced have  been  but  little  studied,  and  certainly  very  little  is 
known  of  them.  The  process  of  "  blowing "  oils,  in  which  at 
a  raised  temperature  air  is  blown  through  the  oil,  results  in  the 
introduction  of  hydrogen  and  hydroxyl  groups  where  there 
are  unsaturated  linkages :  for  the  products  combine  with  more 
acetic  anhydride  than  the  original  oil,  combine  with  less  iodine, 
and  a  part  of  the  acids  acquire  a  higher  molecular  weight.  At 
the  same  time  some  of  the  chains  must  be  broken,  as  volatile 
acids  appear  and  the  saponification  value  is  raised. 

Changes  in  some  respects  similar  to  these  have  been  observed 
by  v.  Fiirth  to  occur  during  germination  in  sunflower  and  castor- 
oil  seeds.  Part  of  the  oil  in  the  seeds  was  used  up,  and  part 
of  what  was  left  was  hydrolysed.  But  the  saponification  value 
(the  amount  of  potash  in  milligrammes  which  is  necessary  for 
neutralising  the  total  fatty  acids  free  and  combined  in  i  g.  of 

1  Duclaux,  Microbiologie^  iv.,  691. 

2  Duclaux,  A.  P.  /.,  1887  ;  Mjoen,  Ch.  Cbl^  ii.,  526,  1897. 


v.]  UNSATURATED  ACIDS  AND  OXY-ACIDS  107 

the  oil)  was  raised,  and  the  mean  molecular  weight  of  the  acids 
therefore  diminished ;  some  of  the  acids  of  high  molecular 
weight  had  broken  down  into  acids  of  lower  molecular  weight. 
And  in  this  process  the  number  of  unsaturated  linkages  and  of 
hydroxyl  groups  had  both  diminished,  indicating  that  these 
are  the  points  in  the  chains  where  the  cleavage  occurs.1 
Changes  of  a  similar  nature  appear  to  occur  in  the  fat  in  the 
body.  In  different  parts  of  the  body  the  fats  certainly  are 
different  in  their  properties.  The  fat  obtained  from  the  heart 
muscle  absorbs,  for  instance,  considerably  more  iodine  than 
that  in  the  connective  tissue.  In  the  sheep  58  per  cent,  as 
compared  with  38  per  cent,  and  in  the  dog  70  per  cent,  as 
compared  with  56  per  cent.  The  fat  obtained  from  the  liver, 
according  to  Rosenfeld,  gives  still  higher  figures. 

It  seems,  therefore,  that  though  the  fat  is  deposited  in 
the  connective  tissues  unchanged,  changes  subsequently  take 
place  in  it,  with  the  result  that  it  contains  more  of  the 
unsaturated  acids  before  it  is  used  in  the  organs  in  which  it  js 
broken  up.  The  unsaturated  linkages  become  more  numerous, 
presumably  because  it  is  at  these  points  that  the  chains  of 
carbon  atoms  are  to  break.  If  we  could  catch  the  process  at  a 
more  advanced  stage,  we  should  find,  as  in  the  plant,  that  some 
of  the  unsaturated  linkages  had  disappeared,  and  the  mean 
molecular  weight  of  the  fatty  acids  had  diminished. 

To  sum  up,  therefore,  on  the  very  scanty  evidence  that  we 
at  present  possess,  we  may  expect  to  find  that  the  fatty  acids 
undergo  oxidation  step  by  step,  each  time  at  the  /3-carbon 
atom  ;  that  an  unsaturated  linkage  is  the  first  move  towards 
this  oxidation,  and  probably  the  formation  of  a  saturated  oxy- 
acid  the  second  ;  the  first  of  these  preparatory  changes  takes 
place  either  in  the  organs  where  the  oxidation  is  carried  out,  or 
before  it  reaches  them  ;  but  after  it  leaves  the  storage  places, 
possibly  in  the  liver. 

These  are  problems  in  animal  metabolism  of  a  fundamental 
nature :  for  the  present,  we  can  hardly  be  more  than  aware  of 
their  existence.  But  the  dim  outlines  must  become  sharply 
1  v.  Fiirth,  H.  £.,  iv.,  430,  1903. 


108  THE  CATABOLISM  OF  FAT  [LECT. 

defined  before  we  can  hope  to  control  or  even  to  follow  the 
course  of  the  chemical  changes  that  constitute  either  health  or 
disease  ;  and  the  first  step  to  this  end  is  to  be  aware  of  them. 

In  the  meanwhile,  however,  quite  unexpected  light  has  been 
thrown  on  one  phase  of  the  catabolism  of  fat  by  the 
physiological  and  pathological  study  of  the  condition  known  as 
acetonuria.  It  has  long  been  known  that  in  diabetes  a  group 
of  substances  occur  in  the  urine  which  are  genetically  related 
to  each  other  :  /3-oxybutyric  acid  by  oxidation  gives  rise  to 
aceto-acetic  acid,  and  this  by  losing  carbonic  acid  to  acetone — 

/H 

CH3 .  C<          .  CH2 .  COOH  —  ->  CH3 .  CO  .  CH0 .  COOH 

\OH  >  CH3 .  CO  .  CH3  +  CO2 

The  first  of  these  changes  is  peculiar  to  the  body ;  the  second, 
which  occurs  probably  in  the  bladder,  is  one  that  can  hardly 
be  prevented,  unless  the  ester  of  aceto-acetic  acid  and  not  the 
free  acid  or  its  salts  be  obtained. 

When  first  these  substances  were  found  in  urine  there 
was  a  tendency  to  associate  them  with  arrested  or  dis- 
ordered sugar  catabolism,  for  it  was  in  the  urine  of  diabetic 
patients  that  they  were  first  recognised.  A  little  later,  however, 
it  was  noticed  that  they  were  excreted  also  under  conditions  in 
which  no  disturbance  of  sugar  metabolism  existed — in  fevers,  in 
starvation,  even  in  healthy  people  on  a  meat  diet ;  and  the 
common  factor  in  all  these  conditions,  diabetes  included,  which 
was  then  believed  to  be  the  cause  of  acetonuria,  was  an  increase 
in  the  rate  at  which  proteids  were  being  broken  down.  This 
theory  also  was  soon  found  to  be  unsatisfactory.  There  is  no 
strict  parallelism  between  the  amount  of  acetone  bodies  excreted 
and  the  rate  of  proteid  destruction.  During  the  first  days 
of  starvation  the  nitrogen  excretion  diminishes,  the  acetonuria 
increases.  In  a  case  of  diabetes,  M.  Levy  recorded  the  excre- 
tion in  the  course  of  three  days  of  342  g.  of  /3-oxybutyric  acid, 
while  the  nitrogen  excreted  corresponded  only  to  262  g.  of 
proteid.1  Similarly,  in  a  case  with  acetonuria,  Satta  found  that 

1  Magnus  Levy,  S.  A.  42,  149,  1899  ;  and,  S.  A.  45,  389,  1901. 


v.]  ACETONURIA  109 

the  variations  in  the  ratio  between  the  amounts  of  sulphuric 
acid  and  acetone  in  the  urine  ranged  from  29  to  0.33,  or  9000 
per  cent.  ;  so  that,  taking  the  sulphuric  acid  as  a  measure  of  the 
proteid  catabolism,  no  kind  of  correspondence  could  be  traced 
between  the  production  of  acetone  and  the  oxidation  of  proteid.1 
And  diabetic  acetonuria  has  been  observed  during  nitrogenous 
equilibrium,  and  even  when  nitrogen  was  being  retained  in  the 
body  and  no  sugar  excreted  in  the  urine.2 

Rosenfield,  who  first  described  the  acetonuria  that  is 
constantly  induced  in  healthy  people  by  restricting  the  diet  so 
as  to  exclude  also  carbohydrates,  found  that  the  excretion  of 
acetone  was  stopped  as  soon  as  the  sugar  or  starch  was  again 
included  in  the  dietary.3  Hirschfeld  found  that  it  was  less 
marked  even  if  the  diet  consisted  only  of  proteids,  but  was  more 
liberally  taken  ;  and  similarly,4  Waldvogel  showed  that  if  in  a 
period  of  starvation  a  day  was  interpolated  on  which  100  g.  of 
proteid  was  taken,  the  amount  of  acetone  excreted  fell 
considerably.5  And  then,  finally,  a  number  of  observers,  first 
among  whom  was  Geelmuyden,  described  an  intensification  of 
acetonuria  as  the  result  of  adding  to  the  amount  of  fat  in  the 
food.6  Thus,  in  an  experiment  on  a  healthy  person  the  dietary 
consisted  of  250  g.  of  butter,  200  g.  of  oil,  and  a  little  wine. 
This  was  taken,  and  nothing  else,  for  five  days.  Intense  acidosis 
came  on ;  diacetic  acid,  /3-oxybutyric  acid,  and  acetone  were 
found  in  the  urine  in  amounts  such  as  occur  only  in  the  severest 
cases  of  diabetes.  On  the  last  day  these  acids  caused  so  much 
of  the  nitrogen  excreted  in  the  urine  to  appear  as  ammonia, 
that  when  the  total  nitrogen  excreted  amounted  to  5.7  g.,  only 
2.7  g.  was  in  the  form  of  urea,  and  as  much  as  2.1  g.  in  the 
form  of  ammonia.7 

1  Satta,  H.  B.  6,  22,  1904.  2  Weintraud,  S.  A.  34,  169,  1894. 

3  Rosenfeld,  M.J.,  p.  467,  1885. 

4  Hirschfeld,  M.  /.,  p.  565,  1895  ;  cf.  Rosenfeld,  CbLf.  1.  M,  1895. 

5  Waldvogel,  M.  /.,  p.  833,  1899. 

6  Geelmuyden,  H.-S.  Z.  23,  431,  1897  ;  and,  26,  381,  1898  ;  and,  41,  128, 
1904. 

7  Landergren,  Sk.  A.  14,  126,  1903  ;  cf.  Gerhardt  and  Schlesinger,  S.  A. 
42,  105,  1899. 


110  THE  CATABOLISM  OF  FAT  [LECT. 

In  practically  all  forms  of  acetonuria,  increasing  the  amount 
of  fat  in  the  food  intensifies  the  condition  :  in  that  due  to  the 
exclusion  of  carbohydrates  from  the  food,  to  diabetes  in  man, 
and  to  phlorrhizine  glycosuria  in  dogs  and  rabbits  kept  other- 
wise without  food. 

Under  certain  conditions,  then,  excess  of  fat  in  the 
food  causes  acetonuria;  under  other  conditions,  in  which  a 
common  feature  is  malnutrition,  in  which,  therefore,  exces- 
sive calls  have  to  be  made  on  the  fat  stored  in  the  body,  the 
excretion  of  this  group  of  substances  related  to  acetone  has 
been  shown  to  be  due  to  the  metabolism  of  fats,  since  it 
has  nothing  to  do  with  that  of  either  carbohydrates  or 
proteids. 

Here,  then,  we  have  evidence  as  to  a  part  of  the  course 
which,  under  certain  conditions  at  any  rate,  the  catabolism  of 
fat  may  take.  Is  this  likely  to  be  the  course  taken  under 
normal  conditions  ?  Traces  of  acetone,  it  is  true,  are  constantly 
found  in  the  breath  and  in  the  urine  of  man.  But  both  men  and 
animals  when  given  /3-oxybutyric  acid,  or  even  aceto-acetic  acid, 
normally  show  no  signs  of  acetonuria  ;  so  that  we  may  assume 
that  these  substances  in  the  normal  organism  are  as  completely 
oxidised  as  the  fats  themselves.  Acetone,  on  the  other  hand,  is 
very  incompletely  oxidised,  the  greater  part  being  excreted  by 
the  lungs  and  kidneys  unchanged.  These  facts  are  compatible 
with  the  hypothesis  that  /3-oxybutyric  is  a  product  of  the  normal 
metabolism  of  fat ;  that,  in  fact,  butyric  acid  undergoes  ^-oxida- 
tion, and  then  gives  rise  to  aceto-acetic  acid,  only  the  aceto- 
acetic  acid  does  not  normally  lead  to  the  formation  of  acetone 
to  any  extent,  but  is  converted  presumably  into  acetic  acid. 
On  this  interpretation  of  the  facts  the  disorder  in  acetonuria 
would  consist  in  the  failure  of  the  organism  to  carry  out  the 
final  steps  in  the  progressive  oxidation  of  the  fatty  acids,  a 
disorder  due  to  the  overtaxing  apparently  of  the  power  of 
carrying  out  the  reaction  repeated  at  each  step  in  the  oxidation 
of  fatty  acids.  The  aceto-acetic  acid  cannot  be  dealt  with,  and 
either  it  or  the  acetone  into  which  it  so  readily  decomposes,  or 
the  substance  immediately  preceding  it  in  the  series  of  deriva- 


v.]  ACETONURIA  HI 

tives  from  the  higher  fatty  acids,  /3-oxybutyric  acid,  is  excreted 
in  the  urine. 

If  we  provisionally  adopt  this  interpretation,  then  all  the 
fatty  acids  in  undergoing  repeated  ^-oxidation  must  give  rise 
to  butyric  acid  ;  each  molecule  of  stearic  or  palmitic  acid  to 
one  molecule  of  butyric  acid.  And  it  may  be  noted  that  the 
experimenters,  who  have  found  that  acetonuria  is  aggravated 
by  the  administration  of  fats  or  fatty  acids,  have  found  that 
it  is  fats  such  as  butter,  in  which  the  mean  molecular  weight 
of  the  acids  present  is  comparatively  low,  that  produce  the 
most  marked  effect;  and  of  the  fatty  acids,  it  is  the  lower 
members  of  the  series,  and  especially  butyric  acid,  that  are 
most  active.1  If  stearic  acid  with  the  molecular  weight  284 
acts  in  this  way  because  it  undergoes  a  process  of  gradual 
oxidative  erosion  until  it  finally  becomes  butyric  acid,  it  is 
clear  that  butyric  acid  with  the  molecular  weight  88  should 

?RA 

be  more  than  three  times  as  active,  since  ~-~^f  or  more  than 

oo 

three  times  as  much  stearic  acid,  must  be  given  to  produce  the 
same  effect.  The  data  at  present  available  are  not,  of  course, 
capable  of  determining  exact  numerical  ratios  of  this  kind. 
And  though  it  may  be  convenient  to  combine  and  co-ordinate 
the  facts  into  some  relationship  to  each  other  and  to  the  general 
problems  of  the  metabolism  of  fat,  such  an  attempt  can  only 
be  made  provisionally  and  for  convenience,  until  the  subject 
has  been  more  investigated.  And  there  are  many  striking 
facts  connected  with  this  subject  which  it  is  difficult  to  com- 
prehend in  any  simple  conception  of  it.  In  the  dog,  for 
instance,  simple  starvation  does  not  bring  on  acetonuria,  as 
it  does  in  man.  And  yet  dogs  treated  with  phlorrhizine,  pro- 
vided that  they  are  not  kept  in  nitrogenous  equilibrium, 
exhibit  the  phenomenon  in  a  marked  degree.2  Then,  again, 
it  is  said  that  oleic  or  erucic  acid  exerts  a  much  less  marked 
influence  than  the  saturated  fatty  acids.3  But,  to  summarise 
our  knowledge  on  this  point  in  human  metabolism,  we  may 

1  Geelmuyden,  H.-S.  Z.  26,  385,  1898  ;  Schwarz,  M.  /.,  p.  976,  1903. 

2  Baer,  S.  A.  51,  271,  1904.  3  Schwarz,  loc.  tit. 


112  THE  CATABOLISM  OF  FAT  [LECT. 

say  that  acetonuria  is  brought  on  by  those  conditions  in  which 
the  fat  of  the  body  or  of  the  food  has  to  be  taken  into  use 
to  an  unusual  extent,  whether  because  the  body  has  lost  the 
power  of  utilising  sugar,  as  in  diabetes,  or  because  there  is  no 
carbohydrate  available. 

In  addition  to  the  substances  from  which  acetone  is  derived, 
sugar,  according  to  a  view  that  has  been  coming  more  and 
more  into  prominence  in  recent  years,  may  also  be  derived 
from  fat  in  the  course  of  metabolism.  It  was  observed  by 
Voit,  and  repeatedly  since  by  others,  that  the  amount  of  oxygen 
taken  up  by  animals,  and  even  man,  could  not  all  be  accounted 
for  in  the  final  products  of  combustion  ;  the  respiratory  quotient 
when  oxidation  is  carried  out  to  completion  cannot  sink  below 
0.7.  If  fats  which  contain  about  1 1  per  cent,  of  oxygen  could 
in  the  course  of  oxidation  give  rise  to  substances  out  of  which 
sugars  containing  more  than  50  per  cent,  of  oxygen  could  be 
formed,  and  these  sugars  stored  in  the  body,  then  clearly 
respiratory  quotients  lower  than  0.7  must  occur.  They  have 
been  observed ;  figures  even  lower  than  0.3  have  been  recorded 
in  hybernating  animals.  Under  these  conditions  the  weight 
of  an  animal  taking  no  food  may,  paradoxically,  rise  steadily 
for  some  time.  This  of  course  does  not  prove  that  sugar  is 
formed  from  fat,  though  it  certainly  shows  that  substances 
richer  in  oxygen  are  being  formed  from  substances  that  are 
poorer,  and  that  the  combustible  material  in  the  body  when 
it  is  oxidised  does  not  suddenly  and  in  one  explosive  reaction 
collapse  into  carbonic  acid,  water  and  the  other  final  products 
of  vital  oxidation.  The  changes  occur  in  stages,  and  though 
usually  the  conditions  permit  of  each  stage  following  its  pre- 
decessor without  interruption,  such  interruption  may  in  certain 
circumstances  occur  at  some  intermediate  stage.  It  is  believed 
that  the  corresponding  phenomenon,  which  was  observed  by 
Wiesner  in  plants  the  seeds  of  which  contain  oil,  and  cause 
during  germination  over  mercury  a  diminution  in  the  volume 
of  the  gas  in  the  vessel  in  which  they  are  contained,  is  correctly 
explained  as  being  due  to  the  formation  of  starch  from  oil.1 
1  Cf.  Seegen,  Pfl.  A.  39,  140,  1886. 


v.]       LOW  VALUES  OF  R.Q. :    HIGH  VALUE  OF  D :  N      113 

But  it  is  only  in  recent  years  that  evidence  has  been  collected 
for  the  view  that  sugar  is  formed  in  the  animal  body  from 
fat.  Some  have  maintained,  especially  Rumpf,  that  the  sugar 
excreted  in  diabetes  is  in  part  derived  from  the  fat  of  the 
body.  Working  under  him,  Hartogh  and  Schumm  have  shown 
that  if  dogs  are  made  to  work  hard  without  food  for  some 
days,  and  then  fed  liberally  on  fat  for  a  week  or  more,  then 
again  made  to  work  and  finally  treated  with  phlorrhizine,  the 
glycosuria  that  is  brought  on  may  become  very  intense.  One 
animal  excreted  as  much  as  145  g.  of  sugar  in  twenty-four 
hours.  It  is  difficult  to  believe  that  after  the  drastic  preparatory 
treatment  sufficient  glycogen  was  left  in  the  tissues  to  produce 
such  quantities  of  sugar  as  they  observed,  and  the  amount  of 
sugar  was  greater  than  could  conceivably  have  been  derived 
from  the  proteid  broken  down  in  the  same  period.  If  every 
carbon  atom  in  the  proteid,  except  those  that  are  excreted  as 
urea,  were  used  for  the  manufacture  of  sugar,  the  sugar  pro- 
duced could  not  weigh  more  than  seven  times  the  amount  of 
nitrogen  excreted.  In  these  experiments  the  sugar  excreted 
in  one  period  of  five  days  amounted  to  as  much  as  nine  times 
the  weight  of  the  nitrogen  found  in  the  urine.1  No  one  can 
suppose  that  even  7  is  an  actually  possible  value  for  the  ratio 
of  the  sugar  derived  from  proteid  to  the  nitrogen  of  that 

proteid.     The  value  found  by  Minkowski  for  this  ratio,  ^,  in 

dogs  after  removal  of  the  pancreas,  was  2.85.  On  the  con- 
stancy of  this  ratio  in  his  experiments  he  bases  his  view  that 
the  sugar  is  derived  in  these  animals  from  the  proteids.  But 
in  human  diabetes,  and  also  in  dogs  after  removal  of  the 
pancreas,  values  even  higher  than  that  found  by  Hartogh  and 
Schumm  have  been  observed — u,  and  even  up  to  12. 2.2  In 
such  cases  it  is  physically  no  less  than  chemically  impossible 
that  proteids  can  have  supplied  the  material  for  the  formation 
of  the  whole  of  the  sugar. 

1  Hartogh  and  Schumm,  S.  A.  45,  17,  I9OI« 

2  Rumpf,   B.  k.    W.,    No.    9,    1899;    Liithje,   D.   A.  f.  k.  M.  80,   98, 
I904. 


114  THE  CATABOLISM  OF  FAT  [LECT. 

Pfltiger1  has  recently  given  strong  expression  to  his  belief 
that,  when  sugar  is  produced  in  the  body  which  cannot  be 
accounted  for  by  the  carbohydrates  of  the  tissues  or  the  food, 
it  must  be  derived  from  the  fats  and  not  from  the  proteids. 
In  an  earlier  paper  he  had  argued  against  all  the  evidence  that 
has  been  adduced  for  the  belief  that  proteids  give  rise  to  sugar 
in  the  course  of  metabolism  ;  showed  that  there  were  many 
flaws  in  this  evidence,  and  that  it  did  not  irrefutably  prove  what 
it  was  designed  to  prove ;  and  finally  concluded,  not  that  the 
conversion  of  proteid  into  sugar  was  not  established,  but  that  it 
never  occurs.  Accepting  this  conclusion,  then,  his  new  experi- 
ments certainly  prove  that  sugar  is  formed  from  the  fats.  He 
removed  the  pancreas  in  dogs  by  Sandmeyer's  operation,2  that 
is  to  say,  excised  the  greater  part,  and  severed  the  connection 
of  the  remainder  with  the  intestine,  a  procedure  which  enables 
the  animals  to  live  for  much  longer  than  they  do  if  the  gland 
is  entirely  extirpated,  and  causes  sooner  or  later  a  glycosuria 
which,  at  first  moderate,  subsequently  becomes  intense.  The 
addition  of  raw  ox-pancreas  to  the  food  of  such  animals 
improves  the  absorption  of  both  fats  and  proteids,  and  at  the 
same  time  greatly  increases  the  glycosuria.  Pfliiger's  dogs 
were  fed  for  several  months  after  the  operation  on  a  preparation 
of  casein  (nutrose)  and  boiled  cod,  the  flesh  of  which  contains, 
in  the  winter  months  when  his  experiments  were  carried  out, 
no  glycogen  and  only  traces  of  fat.  One  of  the  dogs  on  this 
diet  excreted  in  the  course  of  two  months  more  than  3  kg.  of 
sugar.  The  largest  amount  of  glycogen  ever  found  in  a  dog 
amounted,  when  reckoned  as  sugar,  to  4.1  per  cent,  of  the 
animal's  body-weight.  Supposing  that  in  this  experiment  the 
dog,  when  it  was  put  on  the  nutrose  and  boiled  cod  diet,  and 
when  it  weighed  10.3  kg.,  had  this  maximum  amount  of 
glycogen  in  its  tissues,  even  then  422  g.  of  sugar  is  the  most 
that  can  possibly  be  accounted  for  by  the  carbohydrates  of 
its  tissues.  More  than  2\  kilogrammes  of  sugar  must  have 

1  Pfliiger,  Das  Glykogen,  2nd  edit.,  Bonn,  1905  ;  and,  Pfl.  A.  108,  115, 
1905 

*  Z.f.B.  31,  12,  1894. 


v.]  PFLUGER  DERIVES  SUGAR  FROM  FAT  115 

been  produced  from  material  other  than  carbohydrate :  either 
the  proteids  of  the  food  or  the  animal's  own  fat.  Either 
of  these  could  have  supplied  sufficient  carbon ;  for  the  proteid 
food  was  abundant,  and  was  taken  well ;  and  the  fat  in  a  dog 
may  amount  to  46  per  cent,  of  its  weight,  and  on  the  fourth  day 
of  starvation  in  pancreatic  diabetes,  Pfliiger  has  found  as  much 
as  26  per  cent,  of  fat  in  a  dog.  If  all  the  carbon  in  fat  were 
available  for  sugar  formation,  then  100  g.  of  fat  could  yield 
192  g.  of  sugar;  but  according  to  Pfluger's  conception 
of  the  reaction  by  which  fat  yields  sugar  in  the  body,  130  g. 
would  be  formed  from  100  g.  of  fat ;  the  3  kg.  of  sugar, 
therefore,  would  require  2.3  kg.  of  fat,  which  is  not  more 
than  the  dog  weighing  10.3  kg.  may  have  had.  The  dog 
certainly  was  reduced  to  a  skeleton  by  the  end  of  the 
experiment,  and  after  death  had  no  visible  fat  in  its  connective 
tissues. 

During  the  greater  part  of  the  experiment  the  dog  was  in 
nitrogenous  equilibrium,  gaining  about  25  g.  of  nitrogen  in  forty- 
four  days.  But  there  was  no  constant  ratio  between  the  sugar 
and  the  nitrogen  excreted.  For  thirty-four  days  the  average 
value  of  this  ratio  was  about  2.27.  But  it  was  much  lower  than 
this  at  first,  and  fell  off  again  towards  the  end,  several  days  before 
the  power  of  assimilating  proteid  food  showed  signs  of  failing. 
Pfliiger  believes  that  the  sugar  was  derived  from  the  fat  of  the 
body.  And  the  seat  of  this  transformation  he  believes  was 
the  liver.  For  although  the  animal  was  as  wasted  as  if  it  had 
died  of  starvation,  and  had  lost  40  per  cent,  in  weight,  its  liver 
weighed  nevertheless  as  much  as  it  probably  weighed  at  the 
beginning  of  the  experiment.  Like  the  heart  and  the  brain 
in  ordinary  starvation,  he  argues  it  had  been  active  up  to  the 
end ;  and  this  activity  consisted  in  the  conversion  of  fat  into 
sugar,  a  form  of  activity  of  which  the  liver  is  capable,  he  thinks, 
at  all  times.  The  commonly  observed  fact,  that  both  in  men 
and  animals  glycosuria  may  be  increased  by  a  more  abundant 
proteid  diet,  he  explains  as  due  to  the  stimulating  action  exerted 
by  proteids  on  all  the  functions  of  the  liver,  including  this  one 
by  which  fat  is  converted  into  sugar.  Besides  proteids,  other 


116  THE  CATABOLISM  OF  FAT  [LECT 

substances  influence  the  activity  of  this  function  in  the  same 
way — amido  acids,  ammonium  carbonate,  adrenaline,  carbon 
monoxide,  phlorrhizine,  etc. 

In  this  experiment  of  Pfliiger's  one  fact  stands  out  clear 
and  incontestable,  that  sugar  is  synthesised  in  the  body,  and, 
under  the  conditions  observed,  on  a  very  extensive  scale  ;  the 
material  used  in  this  synthesis  can  have  been  derived  only 
from  the  fat  of  the  body  or  the  proteid  of  the  food.  But  if  it 
has  not  been  strictly  proved,  as  Pfluger  maintains,  that  proteids 
ever  supply  what  is  necessary  for  this  synthesis,  neither  does 
this  experiment  positively  prove  that  fat  does  so.  It  proves 
it  only  on  the  assumption  that  proteid  cannot  serve  as  a 
source  of  the  necessary  material,  and  that  has  not  been 
proved. 

It  is  no  easier  to  conceive  a  solution  of  the  problem  presented 
by  the  transformation  of  fat  into  sugar  than  of  the  change  in 
the  opposite  direction,  and  it  cannot  be  more  difficult.  The 
suggestion  made  by  Pfliiger  is  that  the  carbon  atoms  in  stearic 
acid  are  oxidised  one  at  a  time,  and  in  order  beginning  at  the 
terminal  methyl  group,  by  the  intervention  of  ammonia.  When 
the  sixth  carbon  atom  is  reached  the  chain  breaks,  giving  the 
aldehyde  character  to  this  atom  of  carbon,  and  removing  the 
seventh  as  carbonic  acid.  This  process  repeated  leaves  the  four 
terminal  carbon  atoms  in  the  form  of  butyric  acid,  as  is  required 
to  account  for  the  /3-oxybutyric  acid  and  its  derivatives, 
which  are  known  to  arise  in  the  metabolism  of  fat.  Nothing 
analogous  to  this  can  be  quoted,  though  the  final  result  is 
of  course  the  one  needed  for  the  theory.  If  the  fatty  acids 
furnish  material  for  sugar  synthesis  there  would  be  less 
difficulty  in  imagining  some  simple  carbon  compound  being 
formed  in  the  oxidation  of  the  fatty  acids,  and  condensing 
to  give  rise  to  sugar  molecules  some  simple  aldehyde  such  as 
those  that  are  known  to  be  capable  of  such  condensation 
— formic,  for  instance,  or  possibly  glycollic  or  even  glyceric 
aldehyde. 

Before  leaving  the  review  of  the  problems  presented  by  the 
destructive  metabolism  of  fat,  there  are  certain  disorders  to  the 


N 


v.]  FAILURE  OF  FAT  CATABOLISM  117 

study  of  which  we  may  look  as  likely  to  prove  as  important  in 
this  connection  as  the  study  of  diabetes,  in  its  bearing  on  the 
destructive  metabolism  of  sugar.  In  what  is  still  known  as 
fatty  degeneration  we  have  now  learnt  to  see  not  a  degeneration 
in  the  sense  originally  intended,  not,  that  is  to  say,  a  degrada- 
tion of  the  proteids  of  the  cells  resulting  in  their  conversion 
into  fat.  We  have  learnt  that  the  excess  of  fat  in  the  degener- 
ated heart,  as  in  the  degenerated  liver,  is  fat  that  has  been 
imported  from  the  storage  places  for  fat  in  the  connective 
tissues.  The  current  view  of  the  essence  of  the  disorder  in 
such  cases  seems  to  be  that  an  excessive  importation  of  fat 
has  taken  place  ;  that  it  is  not,  therefore,  the  degenerated  organs 
themselves  that  are  primarily  at  fault,  but  the  tissues  which 
should  hold  fat  in  reserve,  or  the  blood  that  transports  it,  which 
have  conspired  together,  as  it  were,  to  dump  fat  in  otherwise 
healthy  organs,  and  so  paralyse  the  normal  traffic  of  those  parts. 
And  yet  it  is  hard,  when  we  find  a  heart  or  liver  the  seat  of 
fatty  degeneration,  to  give  up  the  idea  that  the  disease  has 
struck  at  these  tissues  themselves  in  a  vital  function,  and  it 
is  not  easy  to  assent  to  a  view  that  looks  upon  them  merely 
as  the  victims  of  obesity.  Neither  is  it  necessary.  It  is  more 
compatible  with  our  general  conceptions  of  physiological  pro- 
cesses and  their  disorders  to  suppose  that  since  fat  is  the  most 
valuable  source  of  energy  that  we  have  in  our  bodies,  it  should 
be  made  use  of  in  those  organs  whose  needs  are  greatest  and 
most  insistent.  We  have  seen  that  there  is  much  evidence  that 
fat  is  an  available  source  of  energy  for  the  heart  and  other 
muscles ;  the  more  constant  the  activity  of  a  muscular  organ, 
the  larger  the  amount  of  fat  we  find  in  it.  It  is  not  unreason- 
able, then,  to  suppose  that  it  is  normally  provided  that  there 
should  be  a  regular  system  of  supply  of  fat  from  the  connective 
tissues  by  the  blood-stream  to  such  organs.  But  in  order  to 
balance  this  supply  there  must  be  a  certain  activity  in  the 
chemical  processes  by  which  the  fat  is  made  to  yield  up  its 
energy  in  those  organs.  If  the  chemical  reactions  by  which 
this  result  is  brought  about — the  catabolic  changes  which  liberate 
energy  by  the  demolition  of  fatty  acids — fail,  while  the  supply 


118  THE  CATABOLISM  OF  FAT  [LECT. 

of  fat  by  the  blood-stream  is  maintained,  then  the  result  must 
be  an  accumulation  of  fat — of  imported  fat — in  the  cells  of  the 
tissues  that  normally  use  it  up  as  fast  as  it  is  imported,  but 
now  no  longer  can.  Phosphorus,  or  arsenic,  or  diphtheria 
toxin,  for  instance,  may  act  as  negative  catalysing  agents 
and  retard  the  rate  at  which  a  reaction  normal  to  the 
cells  takes  place,  and  so  by  attacking  a  vital  function  poison 
the  cells  by  robbing  them  of  their  power  of  using  what 
may  in  some  conditions  at  any  rate  be  their  principal  source 
of  energy. 

The  application  of  such  a  conception  as  this  to  the  liver, 
it  is  true,  is  less  obvious.  The  liver  is  the  great  riddle  of  the 
body ;  but  we  do  not  look  to  the  liver  as  to  an  organ  in  which 
transformations  of  energy  are  to  be  most  conspicuous — chemical 
transformations  certainly,  but  changes  involving  in  the  main  but 
little  liberation  of  energy.  It  is  not  likely  that  the  most  valuable 
source  of  energy  in  the  body  should  be  extensively  consumed  in 
the  liver.  If,  therefore,  fat  is  brought  to  the  liver  in  large 
quantities,  as  undoubtedly  appears  to  have  been  the  case  in  livers 
that  are  the  seat  of  fatty  degeneration,  it  is  brought  there  to 
undergo  changes  of  a  different  kind  from  those  that  are  its  fate 
in  a  working  organ  like  the  heart.  It  is  possible,  of  course,  that 
these  changes  involve  reactions  which,  as  well  as  the  quite 
different  reactions  in  the  heart,  are  paralysed  by  the  same 
poisons.  But  it  is  also  possible,  and  perhaps  easier,  to  look  on 
the  accumulation  of  fat  in  the  liver  as  a  kind  of  fat  congestion. 
If  we  suppose  that  the  fat  from  the  connective  tissues  has  to 
undergo  some  preparatory  treatment  in  the  liver  before  it  is 
adapted  to  the  requirements  of  the  organs  in  which  it  is  actually 
oxidised  and  consumed,  then  as  those  organs  no  longer  use  the 
fat  brought  to  them  by  the  blood,  the  blood  will  have  an  in- 
creasing difficulty  in  getting  rid  of  the  fat  that  it  carries  from 
the  liver,  and  be  less  able  to  remove  the  altered  fat  from  the 
liver ;  although,  so  long  as  the  liver  is  able  to  take  up  the 
unaltered  fat  brought  by  the  blood  from  the  connective  tissues, 
the  blood  may  still  be  able  to  carry  the  unaltered  connective 
tissue  fat  There  are  many  conditions  which  are  accompanied 


v.]  FATTY  DISEASE  OF  THE  LIVER  119 

apparently  by  an  increase  in  the  fat  in  the  liver  which  tempt 
one  to  look  on  this  fatty  change  as  a  sign  that  the  stored  fat 
is  being  called  up  at  a  greater  rate  than  usual.  Rosenfeld  has 
especially  studied  the  fatty  liver  that  he  finds  in  dogs  that  have 
been  starved  for  a  week  and  then  treated  for  two  days  with 
phlorrhizine.  The  animals  thus  prepared  are  prevented  by 
their  glycosuria  from  making  use  of  what  is  left  them,  after 
their  week  of  inanition,  of  their  stock  of  carbohydrate,  and  the 
fat  has  to  be  made  use  of.  Pancreatic  diabetes  is  accompanied 
in  the  same  way  by  a  fatty  liver,  and  Pfliiger's  dog  fed  for 
months  on  pure  proteid  food,  having  used  up  almost  all  the 
stored  fat  of  its  body  still  had  a  normal  amount  of  fat  in  its 
liver,  1.7  per  cent  of  the  fresh  organ,  or  11.2  per  cent,  of  the 
dry  substance :  its  last  non-nitrogenous  reserves  had  been 
called  up,  and  were  being  prepared  in  the  liver  for  the  use  of 
the  tissues.  This,  too,  is  how  Pfliiger  regarded  it ;  only  he  goes 
further,  and  considers  the  preparation  of  the  fat  to  consist  in 
its  conversion  into  sugar. 

In  the  discussion  of  the  nature  of  fatty  degeneration 
and  its  bearings  on  the  physiological  problems  of  the  meta- 
bolism of  fat  it  must  not  be  forgotten  that  there  are  certain 
phenomena  included  under  this  general  term  which  require  a 
different  interpretation  from  those  we  have  considered  so  far, 
an  interpretation  which  is  perhaps  not  very  different  from  that 
which  was  in  Virchow's  mind  when  he  introduced  the  term 
fatty  degeneration.  The  nerve  sheaths  do  not  contain  free 
fats,  though  they  contain  complex  combinations  of  higher  fatty 
acids  and  glycerine  with  other  substances.  They  do  not  react 
with  the  histological  reagents  in  common  use  for  staining  fats, 
not  even  with  osmic  acid,  if  by  the  use  of  fixing  agents  they 
are  prevented  from  undergoing  the  changes  which  they  other- 
wise slowly  undergo  in  the  presence  of  osmic  acid.  When  the 
nerves  degenerate  after  division  or  in  disease,  free  fats  appear 
in  them,  and  can  be  at  once  demonstrated  by  any  of  the  re- 
agents for  fats.  In  the  myelin,  therefore,  fat  exists  in  the  form 
of  compounds,  of  the  nature  of  which  we  know  very  little  but  that 


120  THE  CATABOLISM  OF  FAT  [LECT. 

they  do  not  react  like  ordinary  fat.  In  the  heart  and  the  kidney, 
too,  the  considerable  quantities  of  fat  that  can  be  shown  to  be 
contained  in  the  cells  are  normally  combined  in  such  a  way  as 
not  to  give  the  histological  reactions  that  characterise  free  fats. 
It  is  well  known,  too,  that  this  fat  is  but  very  imperfectly 
removed  from  the  dried  and  powdered  tissue  of  these  organs  by 
extraction  with  solvents  for  fats.  In  order  to  extract  the  fat 
completely,  either  digestion  with  pepsin  and  hydrochloric  acid, 
or  boiling  with  alcohol,  or  solution  and  saponification  in  strong 
caustic  alkalies  followed  by  liberation  of  the  fatty  acids 
by  means  of  a  mineral  acid,  must  precede  the  extraction. 
In  the  heart,  however,  which  has  undergone  fatty  degenera- 
tion, as  the  result  for  instance  of  poisoning  by  diphtheria 
toxin,  the  tissue  gives  the  histological  reactions  of  fat,  just 
as  the  degenerated  nerve  does.  And  though  in  the  heart 
there  may  be  some  increase  in  the  amount  of  fat  present, 
and  therefore  there  may  be  a  question  whether  the  histo- 
logically  demonstrable  fatty  change  is  not  all  due  to 
imported  fat  which  is  in  excess,  in  the  degenerated  nerve, 
and,  according  to  Rosenfeld,  also  in  the  degenerated  kidney 
there  is  no  abnormal  amount  of  fat,  and  may  be  even  less 
than  is  normal ;  as  was  found  in  the  case  of  the  "  fatty " 
liver  too,  that  results  from  ligature  of  the  hepatic  artery,  to 
which  reference  was  made  in  the  last  lecture.  In  such  cases 
the  essence  of  the  disorder  is  clearly  not  an  accumulation 
of  imported  fat,  and  must  consist  in  a  change  in  the  mode 
of  combination  of  the  fat.  This  change  we  may  class  with  the 
autolytic  changes  by  which  proteids  are  broken  up  in  cells, 
and,  as  was  pointed  out  in  the  last  lecture,  may  refer  to  as 
degeneration  of  protoplasm,  so  long  as  it  is  clear  that  by  proto- 
plasm is  not  meant  merely  the  proteids  of  the  cells,  but  other 
complex  components  of  living  matter  as  well,  into  the  composi- 
tion of  which,  for  all  that  we  know,  proteids  may  not  enter  in 
any  way  at  all. 

Such  considerations  seem  to  point  to  the  idea  that  the 
catabolic  changes  in  fats  are  brought  about,  not  in  the  free 
simple  glycerides  of  higher  fatty  acids,  but  in  complex  com- 


v.]  FATTY  DEGENERATION  121 

binations  of  these  glycerides  with  other  substances  such  as  we 
know  exist  in  the  medullary  substance  of  nerves,  though  even 
there  we  know  as  yet  only  very  little  concerning  their  exact 
nature  and  constitution,  or  their  relations  to  the  rest  of  the 
substance  which  we  refer  to  as  protoplasm. 


LECTURE    VI 

THE  ASSIMILATION   AND   SYNTHESIS   OF   PROTEIDS 

SINCE  the  first  days  of  modern  physiology,  when  the  group  of 
substances  known  as  proteids  were  first  recognised,  down  to 
comparatively  recent  times,  the  well-known  fact  that  proteids 
are  a  necessary  part  of  the  food  of  all  animals  was  interpreted 
to  mean  that  no  .synthesis  of  proteids  occurred  in  the  animal 
body.  It  was  common  to  draw  this  distinction  between  plants 
and  animals :  plants,  with  hardly  an  exception,  live  without 
proteid  food,  and  must  therefore  make  their  own  proteids  for 
themselves  ;  animals  find  their  proteids  ready-made  in  their 
food,  and  therefore  need  not  and  do  not  make  them.  This 
implied  that  all  the  proteids  in  existence  are  the  product  of 
vegetable  life.  Many  of  the  names  given  to  vegetable  proteids 
show  how  this  implied  conception,  though  not  always  expressed, 
was  always  present :  vegetable  "  fibrin  "  and  vegetable  "  casein  " 
occur  as  well  as  vegetable  "  albumins  "  and  "  globulins,"  names 
given  on  account  of  the  similarity  of  certain  superficial  physical 
properties  of  these  proteids  to  those  of  the  homonymous  sub- 
stances of  animal  origin,  and  with  no  regard  whatever  to 
chemical  constitution.  But  as  the  differences  between  different 
kinds  of  proteids  came  to  be  more  clearly  appreciated,  it  was 
obvious  that  no  proteid  found  in  any  animal  was  the  same  as 
any  proteid  found  in  any  plant ;  and  we  may  now  probably 
go  further,  and  say  that  no  proteid  found  in  any  species  of 
animal  is  identical  with  any  proteid  found  in  any  other  species. 
Even  the  haemoglobin  found  in  different  species  of  animals 


122 


LECT.  vi.]        SPECIFICITY  OF  ANIMAL  PROTEIDS  123 

presents  differences  in  its  properties  which  point  to  differences 
in  constitution.  The  crystal  form,  though  very  generally  similar, 
is  different  in  some  species,  and  the  solubility  even  when  the 
crystal  form  is  the  same,  as  in  man,  the  horse,  and  the  dog, 
may  still  be  very  different.  The  solubility  of  haemoglobin  from 
the  dog  is  given  by  Hoppe-Seyler  as  2  parts  in  100  of  water  at 
5°C.  Dogs'  blood  contains  roughly  from  12  to  15  per  cent, 
and  when  laked,  spontaneously  yields  crystals  of  haemoglobin 
in  the  cold.  Human  blood,  containing  about  the  same  amount, 
treated  in  this  way,  does  not  crystallise.  The  solubility  of 
human  haemoglobin  has  not  been  exactly  determined,  but  is 
certainly  greater  than  that  of  the  haemoglobin  of  dogs'  blood. 
The  analyses  of  haemoglobin  obtained  from  different  species 
also  show  that  the  substance  is  not  one  and  the  same  in  all 
animals.  The  differences  are  probably  confined  to  the  proteid 
part  of  the  molecule,  haematine  being  always  the  same  sub- 
stance, so  far  as  is  known.  Two  atoms  of  sulphur  are  found 
for  one  of  iron  in  the  haemoglobin  of  the  horse,  ox,  and  pig,  but 
three  in  that  of  the  dog. 

The  other  proteids  of  the  blood  also  present  differences  in 
their  properties  in  different  species.  The  serum-albumins  can 
be  crystallised  with  very  different  degrees  of  readiness ;  the 
specific  rotatory  power  of  corresponding  proteids  prepared  in 
the  same  way  from  the  blood  of  different  species  is  notably 
different.  There  is  ground,  too,  for  thinking  that  the  specific 
immunities  and  specific  reactions  of  the  blood  of  different  species 
are  properties  residing  in  the  blood-proteids,  and  depending 
upon  their  chemical  constitution.  The  chemical  differences 
that  have  been  brought  to  light  in  the  composition  of  the  pro- 
tamines  obtained  from  the  sperma  of  different  species  of  fish 
illustrate  the  sort  of  differences  that  may  be  expected  to  exist 
on  a  larger  scale  in  the  larger  molecules  of  the  true  proteids, 
such  as  the  serum-albumins  or  globulins  found  in  different 
species.  But  however  that  may  prove  to  be,  enough  is  already 
positively  known  to  show  that  the  proteids  taken  as  food 
cannot  find  a  place  in  the  economy  of  the  animal  body  till 
they  have  been,  as  it  were,  melted  down  and  recast. 


124     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

At  the  same  time,  our  notions  about  the  meaning  of  the 
changes  undergone  by  proteids  in  digestion  have  been  pro- 
foundly modified.  It  was  formerly  supposed  that  proteids 
being  indiffusible  colloids  could  not  be  absorbed,  until  they 
had  been  converted  into  diffusible  peptones,  and  that  this  was 
the  special  purpose  for  which  digestion  was  necessary.  But.  in 
the  first  place,  it  has  been  shown  that  solutions  of  even  such 
proteids  as  serum-  and  egg-albumin  are  absorbed  from  a  loop 
of  intestine  which  has  been  washed  till  no  trace  of  pepsine  or 
trypsine  can  be  detected  in  the  washings.  These  albumins  are 
among  the  most  typically  indiffusible  members  of  the  class  of 
proteids,  and  yet  they  may  be  absorbed,  if  introduced  into  the 
bowel  in  solution,  although  they  are  not  digested.  In  one  of 
the  experiments  as  much  as  90  per  cent,  was  absorbed,  and  the 
average  extent  of  absorption  was  22  per  cent1  And,  secondly, 
it  must  be  remembered  that  almost  all  the  ordinary  forms  of 
proteid  in  human  food  are  insoluble,  coagulated  in  cooking,  or 
in  the  case  of  the  cereal  proteids,  insoluble  even  if  not  baked 
or  boiled.  The  apparent  exception,  casein,  is  rendered  insoluble 
in  the  stomach.  The  digestive  action  of  the  gastric  and  pan- 
creatic excretions  is,  therefore,  before  all  things  necessary  in 
order  to  convert  these  insoluble  proteids  into  something  soluble, 
and  so  capable  of  passing  into  the  mucous  membrane. 

This  is,  then,  the  first  point  in  which  our  interpretation  of 
the  physiology  of  proteid  digestion  has  been  modified.  The 
second  is,  that  peptones  and  albumoses  are  not  the  only  sub- 
stances formed  from  proteids  in  digestion.  That  this  is  true  of 
the  digestive  action  of  the  pancreatic  ferment,  trypsine,  has  been 
known  for  nearly  forty  years.  But  Kiihne,  who  first  showed 
that  leucine  and  tyrosine  are  formed  when  this  ferment  is  at 
work,2  underestimated  the  extent  of  the  demolition  of  proteids 
effected  by  it.  He  believed  that  one-half  of  the  proteid  mole- 
cule resisted  its  action  entirely.  It  is  now  known  that  this  is 
not  so ;  that  trypsine  can  do  with  proteids  nearly  all  that  boiling 
mineral  acids  can ;  that  all  the  known  products  of  acid  proteo- 

1  Friedlander,  Z./  B.  33,  1896. 

2  Kiihne,  V.  A.  39,  1867. 


vi.]  DIGESTIVE  CHANGES  IN  PROTEIDS  125 

lysis  are  with  very  few  exceptions l  liberated  also  by  trypsine  ;  and 
that,  finally,  nothing  may  be  left,  where  trypsine  has  been  at 
work,  that  can  be  called  in  any  sense  of  the  word  proteid  at  all.2 
But  more  than  this,  we  have  learnt  that  the  action  of  pepsine 
does  not,  either,  stop  with  the  formation  of  peptones.  It  has  been 
found,  for  instance,  that  when  crystalline  serum-albumin  or 
casein  are  digested  with  pepsine  and  hydrochloric  acid,  not  only 
are  substances  containing  nitrogen  formed  which  do  not  give 
the  biuret  reaction,  but  after  so  short  a  time  as  two  hours,  more 
than  50  per  cent,  of  the  nitrogen  originally  contained  in  the 
proteid  may  appear  in  some  form  in  which  it  is  not  precipitated 
by  phosphotungstic  acid — in  a  form,  that  is,  which  is  certainly  not 
proteid,  not  even  peptone — and  this  50  per  cent,  can  include  only 
the  mono-amido  acids,  the  basic  products  presumably  set  free 
at  the  same  time  being  included,  together  with  the  albumoses 
and  peptones,  in  the  precipitate.3  A  considerable  number  of  the 
common  products  of  extreme  proteid  hydrolysis  have  been 
isolated  from  the  fluids  in  which  pepsine  has  been  acting — 
leucine,  amido-valerianic  acid,  aspartic  and  glutamic  acids, 
cystine,  tyrosine,  and  lysine  ;  and  besides  these,  certain  peculiar 
substances  formed  from  these,  in  part  by  the  loss  of  CO2,  as  in 
the  case  of  pentamethylene  diamine  from  lysine,  tetramethylene 
diamine  from  ornithine,  and  oxyphenyl  ethylamine  from  tyrosine, 
and  lastly  leucinimide,  an  anhydride  of  leucine.4  It  is  clear, 
therefore,  that  pepsine  also  can  destroy  to  a  great  extent  the 
proteid  character  of  the  substance  on  which  it  acts. 

But  in  addition  to  what  has  been  recently  learnt  with  regard 
to   the   action   of  trypsine  and  pepsine,  the  discovery  of  the 

1  Cf.  infra,  p.  132. 

2  Kutscher,  H.-S.  Z.  25,  195  ;  26,  no ;  and,  28,  88,  1898  and  1899. 

3  Zunz,  H.-S.  Z.  28,  146,  1899  ;  Pfaundler,  H.-S.  Z.  30,  90,  1900. 

4  Lavroff,  H.-S.  Z.  33,  312,  1901  ;  Langstein,  H.  J5.   I,  507,  and  2,  229, 
1901  and  1902  ;  Salaskin,  H.-S.  Z.  32,  592,  1901.     With  regard  to  the  forma- 
tion of  diamines  from   diamido  acids,  I    have  failed  to  confirm  Lavroff  s 
statement.     The  mucous  membrane  of  dogs'  stomachs  autolysed  with  HC1 
for  two  days  and  filtered,  was  treated  with  crystalline  pure  lysine  bichloride. 
After  two  months,  by  means  of  phenyl  isocyanate,  no  cadaverine  could  be 
detected. 


126     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

ferment,  erepsine,  has  also  contributed  to  the  necessity  for  a 
revision  of  our  views  on  the  meaning  of  proteid  digestion.  This 
ferment  does  not  act  on  all  proteids  alike  :  it  acts  upon  albumoses 
and  peptones,  and  not  upon  other  proteids,  with  the  possible 
exception  of  casein  and  fibrin.1  And  when  it  acts  it  gives  rise 
to  substances  which  do  not  give  the  biuret  reaction,  amongst 
which  tyrosine,  leucine,  lysine,  arginine,  and  histidine  have  been 
identified.  It  is  found  in  the  succus  entericus  ;  so  that  solutions 
of  Witte's  peptone  introduced  into  a  washed  loop  of  intestine 
are  in  an  hour  or  two  transformed  into  solutions  in  which  a  far 
larger  proportion  of  the  nitrogen  escapes  precipitation  with 
tannic  acid  than  was  the  case  before.  But  according  to  Cohn- 
heim  the  amount  of  the  enzyme  found  in  the  mucous  membrane 
itself  is  much  greater  than  that  in  the  succus  entericus,  and  it  is 
mainly  therefore  on  the  albumoses  and  peptones  that  have  been 
absorbed  into  the  intestinal  cells  that  he  supposes  it  to  act.2 
Erepsine,  it  is  true,  is  a  ferment,  on  the  nature  and  even  existence 
of  which  there  is  some  difference  of  opinion.  Cohnheim  demon- 
strated the  presence  of  this  ferment  in  the  intestine  by  washing 
the  mucous  membrane  free  of  pepsine  and  trypsine.  But 
Embden  and  Knoop  deny  that  this  is  possible.  They  found 
that  pieces  of  intestine  obtained  from  normal  animals,  however 
much  they  were  washed,  did  undergo  digestive  changes  when 
kept  at  body  temperature  for  from  one  to  three  hours ;  the 
nitrogenous  substances  that  could  not  be  removed  by  heat- 
coagulation  increased.  But  this  they  ascribed  not  to  the  action 
of  intracellular  ferments,  such  as  erepsine,  but  to  the  presence  of 
trypsine  in  the  recesses  of  the  mucous  surface,  from  which  no 
amount  of  washing  can  remove  it.  For  if,  a  week  before  testing 
the  intestine  in  this  way,  the  pancreatic  ducts  were  ligatured  so 
that  no  trypsine  could  reach  the  gut,  then  these  changes  could 
not  be  detected,  nor  could  any  alteration  be  made  out  in  the 
intensity  of  the  biuret  reaction  due  to  albumoses  or  peptones.3 

1  Lambert,  C.  R.  S.  B.  55,  418,  1903  ;  Embden  and  Knoop,  H.  B.  3,  127  ; 
Kutscher  and  Seemann,  H.-S.  Z.  35,  433,  1902. 

2  Cohnheim,  H.-S.  Z.  35,  136,  and  36,  13,  1902. 
8  Embden  and  Knoop,  H.  B.  3,  120,  1902. 


vi.]  THE  FINAL  PRODUCTS  OF  DIGESTION  127 

It  may  be  questioned  whether  this  necessarily  proves  that  the 
changes  to  be  observed  in  the  normal  intestine  were  due  to 
adherent  trypsine :  for  after  this  operation  the  intestine  was 
abnormal,  not  only  in  containing  no  trypsine,  but  also  in  other 
ways,  and  the  cells  may  not  have  contained  the  enzymes  which 
are  present  in  cells  from  a  normal  intestine. 

Kutscher  and  Seemann  isolated  a  loop  of  gut  by  means  of  a 
Thiry-Vella  fistula,  and  found  that  the  secretion  obtained  from 
the  loop  thirty  days  after  the  operation  had  the  power  of  digest- 
ing deutero-albumose  as  well  as  boiled  fibrin.  But  they  calcu- 
late, by  methods  which  are  not  altogether  unexceptionable,  that 
not  more  than  5  per  cent,  of  all  the  proteid  digested  by  the 
animal  could  possibly  be  accounted  for  by  the  action  of  this 
intestinal  enzyme,  and  conclude  that  erepsine  plays  an  insignifi- 
cant part  in  digestion  in  the  lumen  of  the  bowel ;  and  that  it 
does  not  act  within  the  cells,  they  argue  from  the  fact  that 
during  digestion  they  could  isolate  no  leucine  or  other  crystal- 
line derivative  of  proteid,  either  from  the  portal  blood  or  from 
the  mucous  membrane.  In  their  account  of  proteid  digestion, 
however,  what  erepsine  loses  in  significance  trypsine  gains,  so 
that  the  net  result  is  the  same,  and  they,  like  Cohnheim, 
think  that  the  hydrolysis  of  proteids  in  digestion  is  complete  ; 
the  substances  actually  absorbed  are  not  the  proteids  themselves, 
but  the  simple  crystalline  derivatives,  amido  acids  of  all  kinds.1 

What  is  known,  therefore,  of  the  action  of  each  of  these  three 
ferments,  pepsine,  trypsine,  and  erepsine,  all  points  to  the 
probability  that  the  demolition  of  proteid  molecules  in  digestion 
is  far  more  complete  than  used  to  be  supposed.  It  has  often 
been  argued  that,  although  trypsine  is  capable  of  splitting  off 
amido  acids  from  proteids,  it  does  not  actually  do  so  in  normal 
digestion :  the  grounds  on  which  this  belief  is  generally  based 
are  principally  two.  In  the  first  place,  it  is  said,  if  the  amido 
acids  are  set  free  by  trypsine  in  the  bowel,  it  should  be  possible 
to  find  them  there  when  digestion  is  in  progress.  And  Schmidt 
Miilheim  searched  repeatedly  for  leucine  and  tyrosine  in  the 
intestine  of  dogs  during  digestion,  and  found  only  traces  or 
1  Kutscher  and  Seemann,  ff.-S.  Z.  35,  432,  1902. 


128     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

none.1  MacFadyen,  Nencki,  and  Sieber  also  could  find  none  in 
the  discharge  from  an  intestinal  fistula  in  a  patient  who  had  had 
a  strangulated  hernia  involving  the  ileo-colic  valve.2  But  Kiihne,3 
on  the  other  hand,  obtained  about  3  decigrammes  of  tyrosine  and 
the  same  amount  of  leucine  from  the  duodenum  of  a  dog  into 
which  he  had  introduced,  four  hours  previously,  20  grammes  of 
fibrin.  And  even  earlier  than  that,  Kolliker  and  Miiller  found 
leucine  and  tyrosine  in  the  small  intestine  during  digestion, 
much  more  in  the  upper  parts  than  the  lower,  and  never  any  in 
the  large  intestine.  They  thought  they  came  from  the  pancreatic 
juice,  in  which  they  had  shown  that  these  substances  were 
present.  And  recently  Kutscher  and  Seemann  found  not  only 
leucine  and  tyrosine  constantly,  but  also  lysine  and  arginine. 
It  is  true  that  the  quantities  which  are  obtained  of  these 
substances  are  usually  small ;  but,  then,  if  they  are  formed  they 
are  formed  for  absorption,  and  not  in  order  to  accumulate  in  the 
intestine,  so  that  the  amount  formed  cannot  be  judged  from  the 
amount  left  unabsorbed  in  the  bowel  at  any  particular  moment. 
The  other  principal  ground  on  which  this  belief  is  based  is 
a  teleological  one.  If  proteids  are  necessary  for  the  nutrition 
of  the  body,  it  cannot  be  supposed  that  any  large  quantity  of 
this  indispensable  material  will  be  destroyed  in  digestion. 
Kiihne  himself  regarded  that  part  of  the  proteid  which  was 
broken  down  into  leucine  and  tyrosine  as  so  much  wasted 
proteid.  But  it  is  not  clear  why  we  should  look  upon  leucine 
and  tyrosine  as  wasted  material.  They  are  both  of  them 
when  absorbed  as  completely  oxidised  as  the  proteids  them- 
selves, and,  weight  for  weight,  as  sources  of  energy  they  are 
each  of  them  worth  more  than  proteids.  A  gramme  of  leucine 
yields  on  combustion  6.5  Gal.,  a  gramme  of  tyrosine  5.9.  Even 
glycocoll,  of  all  the  carbon  compounds  which  are  split  off 
from  proteids  the  one  with  the  smallest  molecule,  still  contains 
chemical  energy  equal  to  3  Cal.  per  gramme.  The  energy  con- 
tained in  a  given  weight  of  a  substance  does  not  depend  on 

1  Schmidt  Miilheim,  D.  R.  A.  p.  39,  1879. 

2  Nencki,  MacFadyen,  and  Sieber,  S.  A.  28,  311,  1891. 

3  Kiihne,  V.  A.  39,  155,  1867. 


vi.]         THE  ENERGY  EXCHANGE  IN  PROTEOLYSIS         129 

the  number  of  atoms  aggregated  into  the  molecules  of  the 
substance.  The  other  hydrolytic  changes  effected  in  digestion 
leave  the  sum  of  chemical  energy  practically  unaffected.  The 
energy-value  in  Calories  of  a  gramme-molecule  of  maltose,  cane 
sugar,  or  lactose,  for  instance,  is  in  each  case  just  over  1350  Cal. : 
that  of  the  two  gramme-molecules  of  dextrose  formed  from  one 
of  maltose  is  1347.4:  that  of  the  gramme-molecule  each  of 
glucose  and  fructose  from  cane  sugar  is  1349.6,  and  of  galactose 
and  glucose  from  lactose  is  1343.6.  In  the  two  first  cases  the 
difference  comes  to  about  3  Cal.,  in  the  last  to  nearly  8  Cal, 
Even  this  difference  is  hardly  appreciable.  In  the  hydrolysis 
of  ethyl  butyrate  the  exchange  is  850.1  Cal.  in  the  alcohol  and 
acid  for  851.3  Cal.  in  the  ester  from  which  they  are  derived. 
In  the  case  of  the  proteids  and  the  substances  derived  from 
them  by  hydrolysis,  a  direct  determination  was  in  part  made 
by  Rubner  for  O.  Loewi.1  A  pancreatic  digest,  freed  by  filtra- 
tion from  the  leucine  and  tyrosine  that  had  crystallised  out, 
was  dried  and  the  residue  powdered  :  I  g.  of  the  powder  was 
found  to  yield  4.6  Cal.,  which,  as  Loewi  points  out,  is  only  10 
per  cent,  less  than  the  amount  given  by  I  g.  of  dry  powdered 
meat ;  and  since  considerable  quantities  of  leucine  and  tyrosine 
with  high  heat  equivalents  had  been  removed,  this  fact  must 
account  for  a  great  part  of  this  difference.  But  besides  this, 
the  figure  given  for  meat  is  for  a  mixture  of  chemical  substances, 
in  which  some  fat  at  any  rate  must  have  been  included,  whereas 
the  filtered  acid  pancreatic  digest  must  have  lost  in  addition 
to  most  of  its  leucine  and  tyrosine  all  its  derivatives  of  higher 
fatty  acids.  The  margin  left,  therefore,  for  a  difference  between 
the  energy-value  of  the  proteids  and  that  of  the  substances 
derived  from  them  in  this  experiment  seems  to  be  very  small. 
No  other  more  direct  determinations  of  the  energy  exchange 
in  the  hydrolysis  of  proteids  appear  to  have  been  made.  But 
everything  points  to  its  being  inconsiderable.  It  is  a  mistake, 
therefore,  to  argue  that  if  the  value  of  proteid  food  as  a  source 
of  energy  is  not  to  be  sacrificed,  proteid  digestion  cannot  be 
carried  beyond  the  peptone  stage.  There  is  no  ground  for 

1  Loewi,  S.  A.  48,  328,  1902. 

I 


130     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

thinking  that  changes  that  are  merely  hydrolytic  can  materially 
affect  the  sum  of  energy. 

But,  more  than  this,  we  have  actual  experimental  proof 
that  the  final  products  of  tryptic  digestion,  amongst  which  no 
detectable  trace  of  anything  that  can  be  called  proteid  is  left, 
may  nevertheless  serve  in  the  place  of  proteid  in  the  food  of 
an  animal.  The  first  experiments  establishing  this  were  those 
of  Otto  Loewi.  Dogs  were  fed  on  the  solution  of  pancreas 
obtained  by  allowing  the  glands  to  undergo  complete  autolysis 
in  the  presence  of  chloroform.  This  solution  was  filtered  and 
a  quantity  of  crystalline  products  removed,  and  the  filtrate, 
which  gave  no  trace  of  a  biuret  reaction,  was  administered, 
together  with  starch  and  fat,  to  make  up  the  requisite  sum 
total  of  Calories  in  the  animal's  food.  The  most  successful 
experiment  was  one  in  which  the  dog  took  in  the  course  of 
eleven  days  66.8  g.  of  nitrogen  in  this  form,  and  on  each  day 
the  amount  of  nitrogen  excreted  was  less  than  that  ingested, 
so  that  altogether  9.8  g.  were  retained  in  the  eleven  days,  or  a 
daily  average  of  0.9  g.,  while  the  animal's  weight  went  up 
from  11.9  kg.  to  12.9  kg.1  More  recently  still,  Henriques  and 
Hansen  have  carried  out  similar  experiments  on  rats,  and 
obtained  similar  results.  Animals  were  kept  in  nitrogenous 
equilibrium  for  twelve  to  fourteen  days  while  being  given  no 
proteid,  but  in  place  of  this  the  substances  contained  in  a  digest  of 
ox-pancreas  and  dog's  intestine,  from  which,  under  the  influence 
of  the  enzymes  present,  everything  that  could  give  the  biuret 
reaction  had  disappeared.  But  in  addition  to  this,  two  further 
points  of  no  little  interest  were  determined.  If  the  digested 
fluid  was  precipitated  with  phosphotungstic  acid  and  filtered, 
the  nitrogenous  substances  in  the  filtrate  were  still  sufficient 
to  keep  the  animals  in  nitrogenous  equilibrium.  In  this  case 
the  basic  substances,  and  also  the  polypeptides  according  to 
what  is  at  present  known,  must  have  been  absent  from  the 
food.  In  other  experiments  the  digest  was  treated  repeatedly 
with  96  per  cent,  alcohol  at  50°  C.,  and  so  separated  into  two 
portions,  one  soluble,  the  other  insoluble  in  alcohol ;  and  on 
1  Q.  Loewi,  S.  A.  48,  303,  1902. 


vi.]  NITROGENOUS  EQUILIBRIUM  WITHOUT  PROTEIDS  131 

feeding  the  rats  with  these  it  was  found  that  the  substances 
soluble  in  alcohol  were  efficient  substitutes  for  proteid,  but  not 
those  that  were  insoluble  in  alcohol.  But  though  the  substances 
formed  by  the  hydrolytic  action  of  the  enzymes  of  the  pancreas 
and  intestine  were  shown  under  these  three  different  sets  of 
conditions  to  contain  all  that  is  necessary  for  maintaining 
nitrogenous  equilibrium,  if  the  substances  formed  from  casein 
by  the  hydrolytic  action  of  mineral  acids  were  substituted  for 
these,  then  the  animals  lost  nitrogen,  and  lived  no  longer  than 
others  that  were  fed  on  food  containing  no  nitrogen.1  This 
remarkable  result  has  been  obtained  by  Abderhalden  and 
Rona  as  well,  in  both  mice  and  a  dog.2  There  is  after  all 
something  apparently  that  the  enzymes  leave  intact  but  that 
acids  destroy,  which,  whatever  its  nature  may  be,  is  certainly 
not  in  any  sense  proteid,  but  is  nevertheless  indispensable  in 
the  synthesis  of  proteid.  The  presence  or  absence  of  this  just 
makes  all  the  difference  in  the  food-value  of  the  mixture  of 
nitrogenous  compounds  given  in  these  experiments.  What  is 
the  nature  of  these  essential  substances  which  escape  hydrolysis 
by  trypsine,  but  are  destroyed  by  acids?  To  a  very  great 
extent  the  products  of  hydrolysis  by  enzymes  and  acids  are 
the  same,  and  there  may  have  been  sometimes  a  tendency  to 
assume  that  they  are  throughout  identical.  But  this  is 
improbable.  The  acids  hydrolyse  indiscriminately  almost  all 
that  can  be  hydrolysed — carbohydrates,  for  instance,  as  well  as 
proteids ;  whereas  the  enzymes  are,  as  is  known,  restricted 
within  very  narrow  limits  in  their  action.  To  take  the  familiar 
instances,  the  enzymes  that  hydrolyse  the  polysaccharides  leave 
the  disaccharides  unaltered.  We  are  beginning  to  learn  some- 
thing of  the  hydrolytic  powers  of  trypsine  and  its  limitations 
from  the  experiments  of  Emil  Fischer,  who  has  tested  the 
action  of  this  enzyme  on  definite  synthetic  compounds  of  amido 
acids.  Many  of  these  are  hydrolysed,  the  racemic  ones 
asymmetrically,  while  others  escape  unchanged.  The  factors 

1  Henriques  and  Hansen,  H.-S.  Z.  43,  417,  1905. 

2  Abderhalden  and  Rona,  H.-S.  Z.  42,  530,  and  44,  200,   1904-5.     Cf. 
Henderson  and  Dean,  M.  J.  862,  1903  ;  and,  Lesser,  Z.f.  B.  45,  497. 


132     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

determining  these  different  results  have  already  been  in  part 
indicated  by  Fischer.1 

One  compound  that  resists  the  action  of  trypsine,  but  is 
broken  up  by  acids,  has  been  successfully  traced  by  Fischer  and 
Abderhalden  among  the  products  of  the  pancreatic  digestion 
of  casein  and  other  proteids.  It  was  observed  that  neither 
pyrrholidine  carboxylic  acid  nor  phenyl-alanine  could  be  detected 
in  the  fluid  obtained  by  the  prolonged  digestion  of  casein  with 
trypsine,  unless  the  ester  method  involving  the  exposure  of  the 
substances  to  the  action  of  hydrochloric  acid  was  employed. 
And  the  reason  for  this  was  found  to  be,  that  these  particular 
cleavage  products  were  not  set  free  at  all  by  trypsine,  but  were 
left  combined  with  other  amido  acids  in  a  polypeptide ;  this 
polypeptide  could  be  obtained  by  precipitation  with  phospho- 
tungstic  acid,  was  not  a  peptone,  since  it  did  not  give  the  biuret 
reaction,  but  when  hydrolysed  with  mineral  acids  was  decom- 
posed and  then  yielded  as  much  phenyl-alanine  and  pyrrholidine 
carboxylic  acid  as  the  casein  itself  when  so  treated.2  These 
results,  no  less  than  the  experiments  of  Henriques  and  Hansen, 
show  how  much  we  have  to  learn  about  the  changes  that 
proteids  undergo  in  digestion.  Even  what  we  have  learnt 
shows  more  than  anything  else  how  little  we  know.  For, 
according  to  the  results  obtained  by  Henriques  and  Hansen,  it 
cannot  be  the  polypeptides  that  must  remain  unaltered  in  order 
that  the  products  of  hydrolysis  should  serve  in  the  place  of 
proteids.  For  these  polypeptides  are  precipitated  by  phospho- 
tungstic  acid,  and  the  experiments  expressly  show  that  what 
is  precipitated  by  this  reagent  is  not  essential. 

But  whatever  the  nature  of  the  compounds  may  be  which 
account  for  the  physiological  difference  between  the  products 
of  tryptic  digestion  and  the  products  of  acid  hydrolysis,  it  can 
hardly  be  that  the  substances  spared  by  trypsine  are  the  only 
parts  of  the  proteid  that  are  required  for  proteid  synthesis  in 
the  body.  The  great  bulk  of  the  substances  set  free  in  the 

1  E.  Fischer  and  Bergell,  B.  36,  2592,  1903  ;  and,  B.  37,  3103,   1904 ; 
E.  Fischer  and  Abderhalden,  ff.-S.  Z.  46,  52,  1905. 

2  E.  Fischer  and  Abderhalden,  H.  S.  39,  81  ;  and  40,  215,  1903. 


vi.]          THE  CONDITIONS  OF  PROTEID  SYNTHESIS          133 

hydrolysis  of  proteids  by  enzymes  and  by  acids  are  the  same, 
and  these  substances  enter  into  the  composition  of  the  proteids 
synthesised  in  the  body  in  similar  proportions  to  those  in 
which  they  occur  in  the  proteids  of  the  food.  For  the  present, 
all  we  can  say  is  that  there  appears  to  be  some  kind  of  linkage 
between  certain  groups  in  the  proteid  molecules  which  is  not 
uncoupled  by  the  enzymes  in  the  body,  and  that  when  it  is 
uncoupled,  as  in  acid  hydrolysis,  then  it  is  impossible  for  it  to 
be  coupled  up  again  in  the  body.  This  combination,  which  the 
cells  can  neither  take  to  pieces  nor  put  together  again,  must  be 
present,  in  order  that  the  other  component  parts  of  the  proteid 
molecule  may  gather  about  them  and  group  themselves  round 
them  when  the  synthesis  of  proteids  is  to  occur.  These  con- 
siderations appear  to  suggest  that  the  synthetic  processes  here 
involved  may  be  the  work  of  the  same  agent  as  the  hydrolytic, 
the  limitations  in  its  hydrolytic  power  determining  the  limita- 
tions of  its  synthetic  activity,  as  in  reversible  zymolysis. 

It  is  well  known  that  those  who  have  thought  that  the 
digestive  disintegration  of  proteids  did  not  go  beyond  the 
formation  of  albumoses  and  peptones  have  found  great  difficulty 
in  understanding  how  it  is  that  these  substances  are  not  to  be 
found  in  the  blood.  It  has,  in  fact,  been  long  known  that  they 
are  in  no  small  degree  toxic,  intravenous  injections  affecting 
the  coagulability  of  the  blood,  and  causing  a  considerable  fall 
in  blood-pressure.  In  young  or  feeble  animals  the  injection 
of  o.i  g.  per  kilo  may  be  fatal.1  And  when  the  amount  injected 
does  not  cause  death,  the  albumose  is  removed  from  the  blood 
and  is  excreted  unchanged  in  the  urine.  Spiro  and  Pick,  it  is 
true,  purified  albumoses  in  such  a  way  as  to  do  away  with  their 
toxic  properties,  but  even  so  they  were  excreted  in  the  urine.2 
Neither  can  we  suppose  that  under  normal  conditions  they  are 
taken  up  from  the  portal  blood  by  the  liver,  and  there  altered 
in  such  a  way  as  to  abolish  their  toxicity  and  at  the  same  time 
render  them  available  for  nutrition.  For  Neumeister  showed 
that  they  were  not  to  be  found  in  the  portal  any  more  than 

1  Schmidt  Mulheim,  D.  R.  A.,  p.  30,  1879 ;  Fano,  D.  R.  A.,  p.  277,  1881. 

2  Spiro  and  Pick,  H.-S.  Z.  31,  235,  1900. 


134     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

the  systemic  blood,  and  if  injected  into  the  portal  vein  they 
appear  in  the  urine,  in  spite  of  having  to  pass  first  through  the 
liver.  Nor  can  they  be  found  in  the  lymph  or  the  tissues  any 
more  than  the  blood.1 

There  are  two  well-known  hypotheses  that  have  been 
advanced  to  remove  this  difficulty.  Hofmeister  suggested  that 
the  leucocytes,  which  do  contain  albumose,  might  act  as  carriers, 
take  the  absorbed  albumose  up  and  convey  it  to  the  tissues. 
The  phenomenon  of  post-prandial  leucocytosis  was  quoted  as 
supporting  such  a  conception.  But  Heidenhain  found  that  it 
was  quantitatively  impossible  to  attribute  to  the  leucocytes  such 
a  fundamentally  important  function  in  nutrition.2  And  post- 
prandial leucocytosis,  though  very  commonly  observed,  is  not 
constant,  as  it  should  be,  if  it  were  necessary  for  proteid-absorp- 
tion.  Besides  the  hypothesis  seems  to  require  that  it  should 
be  a  leucocytosis  due  to  an  increase  of  lymphocytes,  which  is 
not  observed  to  be  the  case.  The  explanation,  therefore,  which 
has  most  generally  found  acceptance  is,  that  the  albumoses 
before  they  leave  the  intestinal  epithelium  are  resynthesised 
into  proteids  such  as  are  found  in  the  blood,  albumin  or  globulin. 
This  was  the  explanation  given  of  Salvioli's  often-quoted  experi- 
ments done  in  Ludwig's  laboratory.  He  sent  blood  through 
the  vessels  of  excised  loops  of  intestine  into  which  a  solution 
of  Witte's  peptone  had  been  placed,  and  after  three  or  four 
hours  found  that  peptone  had  disappeared  from  the  bowel,  but 
was  not  to  be  found  in  the  blood.3  Intestinal  perfusion  experi- 
ments which  have  been  carried  out  at  the  Lister  Institute  with 
the  apparatus  devised  by  C.  J.  Martin — by  a  method,  that  is, 
which  is  certainly  an  improvement  on  the  simple  rough  method 
employed  by  Salvioli — have  shown  that  this  is  almost  un- 
questionably not  the  right  interpretation  of  his  results.  It  is 
true  that  the  intestine  under  such  treatment  may  exhibit  move- 
ments for  some  hours,  but  whatever  these  movements  indicate 

1  Neumeister,  Z.f.  B.  27,  315,  1890. 

2  Heidenhain,  Pfl.  A,  43,  suppl.  i,  1888;  Shore,//,  of  Phys.  xi.,  528, 
1890. 

3  Salvioli,  D.  R.  A.,  suppl.  95,  1880. 


vi.]          THE  INTESTINE  AND  PROTEID  SYNTHESIS          135 

as  to  the  condition  of  the  muscular  coat,  they  do  not  necessarily 
indicate  that  the  mucous  membrane  is  living  and  normal. 
The  mucous  membrane  desquamates,  no  absorption  takes 
place,  and  at  the  end  of  the  experiment  the  intestine  is 
distended  with  a  thick  grumous  fluid,  generally  deeply  stained 
with  blood.  In  this  last  respect  Salvioli's  experience  seems  to 
have  been  similar.  The  fluid  recovered  from  the  intestine  is 
found  on  examination,  after  removal  of  coagulable  proteids,  to 
contain  no  less  nitrogen,  sometimes  more,  than  was  introduced 
in  the  peptone  solution.  Peptone  has  disappeared,  it  is  true ; 
for  much  more  of  this  nitrogen  is  in  the  form  of  compounds 
that  are  not  precipitated  by  tannic  acid  than  was  the  case  in 
the  fluid  put  into  the  bowel.  The  peptone  has  disappeared 
owing  to  the  action  of  the  ferments  liberated  from  the  mucous 
membrane,  not  because  it  has  been  absorbed.  And  the  peptone 
is  not  found  in  the  blood,  for  the  simple  reason  that  no  peptone 
has  been  absorbed.1 

Hofmeister  tried  to  obtain  experimental  proof  for  this  same 
hypothesis  in  another  way.  The  mucous  membrane  of  the 
stomach  and  intestines  was  removed  from  animals  killed  during 
digestion,  and  thoroughly  washed.  Parts  of  this  were  boiled 
at  once,  or  heated  merely  to  60°  C.  for  a  short  time,  other  parts 
were  kept  at  the  body  temperature  for  two  or  three  hours. 
Those  portions  which  had  been  heated  at  once  were  found  to 
contain  more  albumose  and  peptone  than  those  the  temperature 
of  which  had  not  been  raised.  The  disappearance  of  albumose 
and  peptone  was  interpreted  as  due  to  the  synthesis  of  coagul- 
able proteid  from  it.2  Neumeister  looked  for  proof  of  the  same 
hypothesis  by  another  method  of  experimentation.  He  floated 
washed  mucous  membrane  taken  from  the  intestine  in  a  solution 
of  peptone  containing  blood  that  was  kept  aerated,  and  found 
that  peptone  disappeared  from  the  solution  and  was  not  to  be 
recovered  from  the  mucous  membrane.3  But  these  results  and 
those  obtained  by  Hofmeister  are  capable  of  another  inter- 

1  Cathcart  and  Leathes, //.  of  Phys.  33,  462,  1906. 

2  Hofmeister,  H.-S.  Z.  6,  69,  1882  ;  and,  S.  A.  19,  20  and  22. 

3  Neumeister,  Z.  f.  B.  27,  315,  1890. 


136     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

pretation,  if  enzymes,  present  either  in  the  cells  or  on  the 
surface  of  the  mucous  membrane,  were  at  work  upon  the 
albumoses,  converting  them  into  substances  of  a  simpler  nature 
that  would  give  no  biuret  reaction.  And  Embden  and  Knoop 
found  that  this  was  so  under  the  conditions  under  which  those 
results  were  obtained.1 

The  hypothesis,  therefore,  that  proteids  are  synthesised  in 
the  intestinal  epithelium  from  albumose  absorbed  during 
digestion,  and  that  these  synthesised  proteids  are  then  passed 
on  to  the  blood,  and  circulate  in  the  blood  for  the  nutrition  of 
the  body,  remains  a  hypothesis.  It  may  appear  to  be  inevitable  ; 
but  no  direct  proof  has  yet  been  advanced. 

If,  however,  we  believe  that  the  changes  undergone  by 
proteids  in  digestion  do  not  stop  with  the  formation  of 
albumoses  or  peptones,  then  a  third  explanation  of  the  absence 
of  these  substances  from  the  blood  may  be  offered.  If  the 
proteolytic  ferments  in  the  intestinal  canal  form  still  simpler 
substances  than  these,  and  if  albumoses  that  are  absorbed  into 
the  epithelium  are  there  acted  on  by  erepsine,  that  which  reaches 
the  blood  in  proteid  absorption  may  no  longer  be  proteid  at 
all.  It  may  be  amido  acids  and  similar  final  products  of 
hydrolysis  that  are  circulated  and  supply  the  body  with 
what  is  necessary  for  the  restitution  processes  of  metabolism 
and  for  growth.  But  if  so,  it  should  be  possible  to  detect  these 
substances  in  the  blood  ;  and  the  problem  that  now  confronts 
us  is  to  determine  whether  this  can  be  done,  more  especially 
during  the  absorption  of  proteids. 

But,  first  of  all,  we  may  do  well  to  consider  how  much 
nitrogen  in  such  forms  we  can  expect  to  find,  even  supposing 
that  the  whole  of  the  proteid  in  the  food  reaches  the  blood  in 
the  form  of  simple  final  products  of  hydrolysis,  and  as  such  is 
conveyed  to  the  tissues  where  it  is  required.  A  man  takes 
100  g.  of  proteid  in  the  day,  we  will  suppose,  containing  about 
16  g.  of  nitrogen.  Absorption  begins  soon  after  the  first  meal, 
and  lasts  with  little  if  any  interruption  till  four  or  five  hours 
after  the  last  meal.  The  average  rate  of  absorption,  therefore, 
1  Embden  and  Knoop,  //.  B.  3,  128,  1902. 


vi.]   THE  SEARCH  FOR  AMIDO  ACIDS  IN  THE  BLOOD  137 

of  the  1 6  g.  of  nitrogen  cannot  be  much  more  than  about  i  g. 
an  hour.  The  circulation  time  of  the  intestine  is  short,  almost 
as  short  as  that  of  the  lungs,  and  is  probably  much  less  than  a 
minute,  perhaps  not  more  than  half  a  minute ;  and,  therefore,  if 
there  is  one-tenth  of  the  blood,  or  about  half  a  litre,  in  the 
intestine  during  absorption  of  food — and  there  is  probably  more 
than  this — half  a  litre  of  blood  passes  through  the  intestine 
every  minute ;  that  is  to  say,  30  litres  in  an  hour  at  the  lowest 
computation,  or  60  litres  by  what  is  probably  a  fairer  mode  of 
reckoning.  Thirty  to  sixty  litres  are  available,  therefore,  for 
the  removal  of  I  g.  of  nitrogen  corresponding  to  6.25  g. 
of  proteid.1  This  amount  of  nitrogen  by  the  hypothesis  is  dis- 
tributed over  a  large  number  of  substances  formed  by  hydrolysis 
from  the  proteid,  and  is  not  present  all  of  it  in  one  form. 
Leucine  is  probably  the  most  abundant  of  any  of  these 
substances,  and  if  we.  take  20  per  cent,  for  the  amount  of 
leucine  obtained  from  the  proteid,  the  amount  of  leucine  in  the 
30  to  60  litres  of  blood  would  be  1.25  g.,  or  in  I  litre  20  to  40  mg. 
To  detect  this  amount  of  leucine  in  blood,  which  contains 
some  20  per  cent,  of  proteids,  may  be  possible,  but  it  cannot 
be  easy.  Tyrosine,  of  which  proteids  generally  contain  less  than 
5  per  cent,  would  be  present  in  less  than  a  quarter  of  this 
amount;  and  the  other  cleavage  products,  also  obtainable  from 
proteids  in  small  amounts,  one  may  well  abandon  all  hope  of 
isolating  in  a  recognisable  form,  since  they  are  certainly  less  easy 
to  identify  than  these.  Kutscher  and  Seemann,  however,  tried 
to  isolate  crystalline  cleavage  products  of  proteids  from  the 
portal  blood  of  dogs  during  proteid  absorption,  and  they 
failed  in  every  case.  Their  procedure  was  to  give  dogs  500  g. 
of  meat,  that  is  about  100  g.  of  proteid,  at  about  two  o'clock  in 
the  afternoon,  and  again  another  100  g.  about  ten  at  night, 
and  the  next  morning  at  ten  to  bleed  from  the  portal  vein. 

1  Cybulski  (S.  A.  37,  p.  39,  1895)  measured  the  rate  of  flow  through  the 
portal  vein  of  a,  dog  weighing  9.5  ko.,  and  found  it  to  be  on  an  average 
9090  c.c.  per  hour,  or  about  150  c.c.  per  minute.  In  a  man  of  eight  times 
the  weight  of  this  dog,  at  the  same  rate  the  flow  would  be  1200  c.c.  per 
minute,  or  72  litres  per  hour. 


138     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

After  coagulating  the  proteids  by  heat,  the  filtrate  was 
evaporated,  to  allow  leucme  or  other  recognisable  substances  to 
crystallise  out ;  no  crystals  of  this  nature  were  in  any  case 
detected.  On  one  occasion,  in  a  dog  that  six  hours  previously 
had  had  a  large  meal  of  meat,  they  connected  the  portal  vein 
with  the  vena  cava  by  means  of  an  oiled  tube,  and  then  tied  the 
aorta  below  the  origin  of  the  superior  mesenteric  branch,  and 
also  the  subclavian  and  carotid  arteries  close  to  their  origin,  and 
the  renal  vessels  on  both  sides,  so  that  the  blood  then  circulated 
through  the  intestines,  heart,  and  lungs,  while  the  liver  and 
nearly  the  whole  of  the  rest  of  the  body  was  excluded  from  the 
circulation.  With  the  help  of  artificial  respiration,  the  heart 
was  kept  beating  for  about  an  hour,  but  at  the  end  of  that 
time  no  amido  acids  could  be  isolated  from  the  blood.  The 
conclusion  to  which  they  came  was  that  these  substances,  which 
they  believe  to  be  the  principal  products  of  proteid  digestion, 
are  built  up  in  some  way  into  proteid  in  the  intestine,  just  as 
Ludwig  and  Salvioli,  Hofmeister  and  Neumeister  had  supposed 
that  the  albumoses  and  peptones  are.  In  support  of  this  conclu- 
sion they  found  that  a  substance  could  be  extracted  from  the 
mucous  membrane,  which  is  not  precipitated  by  phosphotungstic 
acid,  but  gives,  after  boiling  with  sulphuric  acid,  leucine  crystals 
on  evaporation.  This  they  regarded  as  a  synthetic  product 
formed  from  the  absorbed  amido  acids  in  the  mucous  membrane.1 
The  results  of  their  experiments,  though  they  are  compatible 
with  their  conclusion,  do  not  prove  it ;  for  they  are  equally 
compatible  with  other  hypotheses  different  from  theirs ;  for 
instance,  that  the  absorbed  amido  acids  undergo  other  changes 
in  the  process  of  absorption  not  necessarily  of  a  synthetic 
nature,  or  that  they  are  removed  from  the  blood  by  the  tissue 
cells  as  quickly  as  they  enter  it. 

Leaving  on  one  side  for  the  present  any  other  changes  that 
there  may  be  ground  for  believing  the  amido  acids  may  undergo 
in  the  intestine  or  liver,2  and  supposing,  as  is  after  all  probable, 
that  the  products  of  digestive  hydrolysis  of  proteids  that  enter 

1  Kutscher  and  Seemann,  H.-S.  Z.  34,  528,  1902. 

2  Cf.  infra. 


vi.]  AMIDO  ACIDS  FOUND  IN  THE  BLOOD  139 

the  blood  unchanged  are  taken  up  by  the  tissue  cells  at  a  rate 
not  appreciably  different  from  the  rate  at  which  they  are 
absorbed  by  the  blood  from  the  intestine,  then,  as  we  saw  from 
the  comparison  of  the  blood  flow  with  the  rate  of  absorption,  the 
amount  of  any  one  cleavage  product  present  in  the  blood  at 
any  moment  may  be  too  small  for  the  isolation  of  crystals  of 
this  substance  to  be  practicable.  But  it  might  still  be  possible 
to  detect  by  some  test  for  amido  acids  in  general  an  increase  in 
the  sum  total  of  these  bodies  in  the  blood  during  proteid 
absorption.  The  most  delicate  reagent  for  amido  acids  is  the 
chloride  of  naphthalene  sulphonic  acid,  which  reacts  with  the 
amido  group,  just  as  benzoyl  chloride  does  when  together  with 
glycocoll  it  forms  hippuric  acid,  the  products  that  are  formed 
being  insoluble  and  crystallising  well.1  By  the  use  of  this 
reagent  the  presence  of  glycocoll  has  been  detected  in  the  urine 
of  rabbits  poisoned  with  phosphorus,  and  glycocoll  is,  owing  to 
its  solubilities,  exceedingly  difficult  to  trace  in  fluids  that  contain 
only  traces  of  it.  v.  Bergmann  used  this  acid  chloride  to  test  for 
the  presence  of  amido  acids  in  the  blood  from  a  case  of  acute 
yellow  atrophy  of  the  liver.  After  coagulating  by  heat  the 
proteids  in  270  c.c.  of  blood  the  filtrate  gave  a  precipitate  with 
the  sulphochloride  amounting  to  more  than  2  g. ;  but  actual 
isolation  of  the  component  substances,  which  were  in  part 
crystalline,  proved  to  be  impossible.  So,  too,  with  the  blood  of 
dogs  killed  during  digestion,  precipitates  were  obtained,  though 
the  identification  of  any  single  component  was  not  practicable.2 

It  is  clear  that  if  it  were  possible  to  prevent  the  removal  by 
the  tissues  of  any  cleavage  products  of  proteids  that  are  absorbed 
into  the  blood,  our  chance  of  finding  them  in  the  blood  would  be 
increased.  Cohnheim  has  for  this  purpose  made  experiments  on 
the  intestines  of  Octopus  vulgaris  and  Eledone  moschata,  in 
which  animals  the  intestine  floats  as  it  were  in  the  blood.  He 
introduced  a  solution  of  peptone  into  the  excised  and  ligatured 
intestines,  and  floated  them  in  blood,  which  he  kept  oxygenated  ; 

1  Fischer  and  Bergell,  JB.  35,   3779,   19°2  5    Abderhalden  and  Bergell, 
H.-S.  Z.  39,  u,  1903. 

2  v.  Bergmann,  H.  B.  6,  40,  1904. 


140     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS  [LECT. 

at  the  end  of  twenty  hours  he  found  that  the  blood  contained 
lysine,  arginine,  leucine,  and  tyrosine,  whereas  the  blood  during 
digestion  in  the  living  animal  contained  none  of  these  sub- 
stances that  could  be  detected.1 

In  some  experiments  carried  out  at  the  Lister  Institute  on 
this  point  the  attempt  was  made  in  the  first  instance  to  obtain 
the  absorbed  products  in  a  greater  concentration  by  allowing  the 
blood  to  circulate  only  through  the  intestine,  using  Martin's 
perfusion  apparatus ;  but,  as  mentioned  above,  it  has  proved  so 
far  impossible  to  keep  the  mucous  membrane  in  the  excised 
intestine  of  dogs  in  such  a  condition  that  it  should  absorb  any 
nitrogenous  substances  at  all.  The  appearances  suggest  that 
the  mucous  membrane  perishes  through  auto-intoxication.  If 
only  it  should  be  possible  to  overcome  this  difficulty,  it  should 
be  much  easier  to  detect  an  increase  of  amido  acids  in  the  blood 
in  this  way  than  in  the  entire  animal.  But  even  in  the  entire 
animal,  with  a  solution  of  peptone,  of  albumose,  or  of  the  final 
products  of  tryptic  digestion  introduced  into  the  bowel  between 
ligatures,  when  absorption  takes  place,  as  is  the  rule,  there  is 
quite  constantly  on  examining  the  blood  an  increase  in  the 
nitrogen,  in  the  form  of  compounds  which  are  not  precipitated 
by  tannic  acid.  The  nature  of  these  compounds  of  nitrogen 
added  to  the  blood  during  absorption  has  not  been  determined, 
but  they  cannot  be  proteid,  not  even  albumose.  It  has  been 
shown  that  the  very  small  increase  in  the  ammonia  in  the  blood 
is  by  far  not  sufficient  to  account  for  them.  But  urea,  on  the 
other  hand,  appears  to  account  for  one  half,  while  the  rest  almost 
certainly  must  be  in  the  form  of  amido  acids  and  similar  sub- 
stances.2 

The  whole  question  of  the  absorption  of  nitrogenous  foods, 
and  as  to  the  nature  of  the  material  used  in  the  synthesis  of 
proteids  in  the  animal  body,  is,  however,  still  further  complicated 
by  the  fact  that  it  has  recently  been  shown  in  more  than  one 
laboratory  that,  occasionally  at  any  rate,  albumoses  may  be 
present  in  the  blood.  Embden  and  Knoop  found  this  to  be  the 

1  O.  Cohnheim,  H.-S.  Z.  35,  407,  1902. 

2  Cathcart  and  Leathes,  //.  of  Pkys.  33,  462,  1906. 


vi.]  ALBUMOSE  IN  THE  BLOOD  141 

case  repeatedly  ;  but  since  in  blood  from  animals  killed  during 
absorption  of  proteids  they  sometimes  failed  to  find  albumose, 
whereas  in  blood  from  starving  animals  they  sometimes  had 
positive  results,  they  are  unwilling  to  draw  any  conclusions  as  to 
the  significance  of  their  results.1  So,  too,  v.  Bergmann  and 
Langstein  found  that  after  removing  coagulable  proteid  from  the 
blood  plasma  of  dogs  killed  during  digestion  there  was  still  a 
considerable  portion  of  the  total  nitrogen  of  the  plasma  to  be 
found  in  the  filtrate,  in  one  case  as  much  as  14.7  per  cent,  and 
some  of  this  was  in  one  instance  shown  to  be  in  the  form  of 
primary  albumose.  Human  blood  also  has  occasionally  been 
found  to  contain  albumose.2 

It  may  be  that  none  of  these  recorded  observations  of  the 
presence  of  albumose  in  the  blood  have  any  direct  bearing  on 
the  question  with  which  we  are  concerned  here.  But  they 
throw  some  doubt,  especially  those  in  the  last  paper  referred  to, 
on  the  conclusiveness  of  the  results  which  have  been  hitherto  the 
foundation  of  our  conceptions  of  proteid  absorption. 

The  situation  may  be  summed  up  in  a  few  words.  Till 
recently,  it  was  believed  that  proteids  were  absorbed  mainly  if 
not  entirely  as  albumoses  and  peptones ;  that  these  substances 
were  converted  by  a  synthetic  change  carried  out  in  the 
intestinal  mucous  membrane  into  the  coagulable  proteids  found 
in  the  blood  ;  that  these  blood  proteids  supplied  the  needs  of  the 
body,  and  were  the  material  used  for  all  tissue  repair.  We  have 
gradually  learnt  that  the  first  of  these  articles  of  belief  requires 
considerable  modification,  we  have  to  recognise  that  the  second 
remains  purely  hypothetical,  and  that,  therefore,  the  third  is  little 
if  anything  more  than  a  preconception. 

The  problem  as  it  presents  itself  to  us  now,  is  rather  this  :  is 
the  synthesis  of  proteid,  which  is  so  important  a  factor  in  the 
metabolism  of  all  growing  and  living  animals,  a  function  only  of 
the  intestinal  epithelium  ?  Direct  evidence  is  not  as  yet  forth- 
coming, and  we  must  be  content  with  some  working  hypothesis 
put  together  from  general  physiological  and  biological  considera- 

1  Embden  and  Knoop,  H.  B.  3,  120,  1902. 

2  Bergmann  and  Langstein,  H.  B.  6,  37,  1904. 


142     ASSIMILATION  AND  SYNTHESIS  OF  PROTEIDS   [LECT. 

tions.  But  the  result  of  attempting  to  form  such  a  hypothesis 
at  the  present  time  will  be  somewhat  different  from  what  it  was 
a  few  years  ago.  We  know  that  in  the  seeds  of  plants  proteids 
are  often  stored  in  considerable  quantity  as  food  stuff  for  the 
growing  seedling.  During  germination  these  proteids  are  hydro- 
lysed,  and  circulated  in  the  sap  in  the  form  of  the  familiar 
cleavage  products.  Schultze  and  Winterstein  have  isolated  from 
the  seedlings  of  various  species  of  plants  a  long  list  of  these 
cleavage  products :  leucine,  iso-leucine,  amido-valerianic  acid, 
alanine,  glutamic  acid,  aspartic  acid,  phenyl-alanine,  tyrosine,  pyr- 
rholidine  carboxylic  acid,  cystine,  tryptophane,  lysine,  arginine, 
histidine.  All  these  substances  can  be  formed  during  digestion 
in  animals.  In  the  plant  it  is  in  these  forms  that  the  nitro- 
genous material  is  supplied  to  the  cells  during  the  period  of 
most  active  growth,  and  from  these  unquestionably  the  proteids 
are  synthesised.  In  animals,  till  recently  we  have  believed  that 
the  intestine  synthesised  from  these  or  more  complex  substances 
the  serum-albumin  and  globulin  found  in  the  blood,  and  that  it 
was  with  these  highly  organised  coagulable  proteids  that  the 
cells  of  the  body  were  actually  nourished.  No  account  has  been 
commonly  taken  of  the  fact  that  these  proteids  of  the  blood 
must  be  taken  to  pieces  and  again  put  together,  rearranged  on  a 
different  plan,  if  they  are  to  serve  for  the  making  of  proteids 
and  nucleo-proteids  in  the  cells  of  the  muscles  and  other  organs 
in  which  the  destructive  changes  of  life  are  felt.  The  proteids 
circulating  in  the  blood  are  a  currency  which  is  not  legal  tender. 
And  no  account  has  been  commonly  taken  of  the  familiar  fact 
that  when  no  food  is  obtainable,  certain  organs  maintain  for 
themselves  a  normal  composition  at  the  expense  of  the  substance 
of  other  organs.  When  the  spleen,  liver,  or  the  muscles  of  the 
limbs  dissolve  away  in  starvation,  the  heart  feeds  on  what  they 
supply.  Are  the  proteids  of  these  organs  converted  into  serum- 
albumin  and  globulin,  or  are  they  melted  down  by  autolytic 
processes  into  the  same  cleavage  products  as  are  formed  in  the 
digestion  of  food,  and  in  this  form  thrown  into  the  circulating 
blood,  which  is  thus  in  a  position  to  supply  the  heart  and 
diaphragm  with  just  what  they  are  accustomed  to  receive  in  the 


vi.J  AS  IT  APPEARS  TO  US  NOW  143 

blood  from  the  digestive  organs?  In  the  equally  familiar  and 
often  quoted  marvel  of  animal  metabolism,  the  salmon  in  fresh 
water,  when  the  flesh  of  its  own  muscles  takes  the  place  of  food 
and  supplies  the  fish  with  what  is  necessary  for  the  development 
of  the  sexual  glands,  the  synthesis  of  proteids,  of  a  kind  pro- 
foundly different  from  those  that  supply  the  material  for  this 
synthesis,  must  be  effected  out  of  fragments  of  the  original 
molecules  rearranged  and  put  together  in  new  combinations,  by 
processes  in  which  the  intestine  can  hardly  be  supposed  to  play 
a  part.  And  what  applies  to  the  salmon  or  the  starving  animal 
applies  also  to  patients  in  acute  fevers,  for  instance,  or  in 
hysterical  or  other  conditions  in  which  insufficient  food  or  none 
at  all  is  taken.  The  hypothesis  to  which  such  considerations  as 
these  are  leading  us  is  that  the  synthesis  of  proteids  is  a  function 
of  every  cell  in  the  body,  each  one  for  itself,  and  that  the 
material  out  of  which  all  proteids  in  the  body  are  made  is  not 
proteid  in  any  form,  but  the  fragments  derived  from  proteids  by 
hydrolysis,  probably  the  amido  acids,  which  in  different  combina- 
tions and  different  proportions  are  found  in  all  proteids,  and 
into  which  they  are  all  resolved  by  the  processes,  autolytic  or 
digestive,  which  can  be  carried  out  in  every  cell  in  the  body. 


LECTURE  VII 

PROTEID   CATABOLISM 

THE  catabolism  of  proteids  in  the  body  results  in  the  discharge 
of  most  of  the  nitrogen,  commonly  not  far  short  of  90  per  cent, 
in  the  form  of  urea.  From  100  g.  of  proteid  about  30  g.  of  urea 
are  formed,  containing  only  6  g.  of  carbon  out  of  the  52  or  more 
contained  in  the  proteid.  So  that  nearly  90  per  cent,  of  this 
carbon  is  disposed  of  in  other  ways,  and  finally  leaves  the  body 
altogether  dissociated  from  the  nitrogen,  as  carbonic  acid.  In 
the  study  of  proteid  catabolism  we  have  to  account,  therefore, 
for  the  formation  of  urea  and  of  any  other  constant  nitrogenous 
excretory  products,  but  also  for  the  disposal  of  the  greater  part 
of  the  carbon  of  the  proteids,  and  at  the  same  time  to  form 
some  conception  as  to  how  far  these  two  sides  of  proteid 
catabolism  are  associated  together  ;  whether,  that  is,  the  carbon 
and  nitrogen  are  separated  early  or  late  in  the  series  of  changes 
which  end  with  the  production  of  little  but  urea,  carbonic  acid, 
and  water. 

It  is  customary  in  discussing  proteid  catabolism  to  work 
back  from  these  end-products,  or  more  generally  only  those  that 
contain  nitrogen,  and,  by  trying  to  give  an  account  of  the 
manner  in  which  they  come  into  being,  to  aim  at  getting  in  this 
way  a  general  view  of  the  destructive  changes  to  which  proteids 
are  submitted  in  the  body.  It  is  possible  that  this  general  view 
will  be  more  easily  attained  by  reversing  this  process,  starting 
from  the  proteids  themselves  and  working  down  to  the  end- 
products,  not  omitting  the  carbonic  acid.  As  soon  as  sufficient 

144 


LECT.  vii.]          INTRACELLULAR  PROTEOLYSIS  145 

data  have  been  collected,  this  must  certainly  be  the  order  of 
procedure,  and  even  now  it  may  be  worth  while  seeing  how  far 
it  is  possible  to  follow  it. 

The  catabolism  of  proteids  takes  place  within  the  cells  of 
the  various  tissues.  And  one  of  the  most  outstanding  facts 
among  those  that  have  come  to  light  in  recent  years  is,  that  the 
cells  of  almost  all  the  organs  of  the  body  have  been  shown  to 
contain  enzymes  that  hydrolyse  proteids — have  within  them,  that 
is,  the  means  of  taking  to  pieces  those  complex  condensation 
products  of  simple  and  compound  amido  acids  which  are  known 
as  proteids,  almost,  if  not  quite  as  completely  as  strong  boiling 
mineral  acids.  Salkowski,1  about  fifteen  years  ago,  was  the  first 
to  show  that,  like  the  unicellular  yeast  plants,  the  liver  and 
muscles  of  dogs,  kept  at  body-temperature  in  the  presence  of 
antiseptics,  underwent  changes,  to  which  he  gave  the  name  of 
auto-digestion,  and  that  in  those  changes  leucine,  tyrosine,  and 
other  soluble  nitrogenous  substances  were  produced  at  the 
expense  of  the  proteids.  Jacoby2  extended  this  study,  and 
introduced  the  word  autolysis,  which  has  been  generally 
adopted  as  the  name  for  such  changes.  He  showed  that  the 
excised  liver  gradually  undergoes  liquefaction,  and  in  the 
process,  besides  leucine,  tyrosine,  glycocoll,  and  tryptophane, 
ammonia  and  other  basic  substances  are  formed  ;  and  also  that 
the  autolysis  is  far  more  rapid  if  aseptic  precautions  render  the 
use  of  antiseptics  unnecessary.  The  enzyme  which  effected 
this  could  be  precipitated  with  ammonium  sulphate,  and  a 
solution  of  the  precipitate  retained  its  power  of  setting  up  these 
changes.  In  the  spleen,  thymus,  lymphatic  glands,  kidneys, 
and  heart,  Hedin 3  found  that  similar  autolytic  processes 
could  be  traced ;  and  the  enzymes  separated  and  distinguished 
from  each  other ;  while  the  study  of  the  substances  formed 
showed  that  the  disintegration  of  proteids  is  as  complete  as 

1  Salkowski,  D.  R.  A.,  1890;  Z.f.  k.  M.,  1890;  Schwiening,  V.  A.  136, 
1894  ;  and,  Biondi,  V.  A.  144,  1896. 

2  Jacoby,  H.-S.  Z.  30,  149,  1900  ;  cf.  Conradi,  H.  B.  i,  144,  1902. 

3  Hedin  and  Rowland,  H.-S.  Z.  32,  341  and  531,  1901  ;  and,  Hedin,//. 
ofPhys.  30,  155,  1903. 

K 


146  PROTEID   CATABOLISM  [LECT. 

in  the  action  of  the  ferments  secreted  by  the  digestive 
glands.1 

These  autolytic  processes  are,  it  is  true,  frequently  referred 
to  as  if  they  were  merely  death  changes,  and  as  if  they  corre- 
sponded to  nothing  that  goes  on  during  the  life  of  the  cells. 
And  the  discussion  that  was  raised  and  so  long  kept  up  over 
Claude  Bernard's  discovery  of  the  ferment  in  the  liver  that 
hydrolyses  glycogen,  has  threatened  to  come  up  again  over  the 
proteolytic  enzymes  of  the  cells.  The  exact  part  played  by 
them  during  life  in  the  cells  does  not  lend  itself  to  investigation, 
perhaps,  but  it  is  certainly  easier  to  believe  that  they  do  operate 
in  the  metabolism  of  the  living  cell,  controlled  and  checked  by 
the  conditions  that  balance  during  life  the  tendencies  to 
chemical  reaction  one  against  the  other,  than  to  suppose,  as  is 
otherwise  implied,  that  every  cell  is  furnished,  as  it  were,  with 
the  means  of  putting  an  end  to  itself,  to  which  recourse  is  to  be 
had  under  no  circumstances  till  the  worst  comes,  and  life  is  no 
longer  possible.  For,  that  they  are  not  merely  escaped  trypsine, 
as  Neumeister  suggested,  was  shown  by  the  fact  that  extirpation 
of  the  pancreas  does  not  cause  them  to  disappear.2 

They  have  been  shown  to  be  concerned  in  the  removal  of 
pneumonic  exudation,  since,  while  normal  lung  and  lung  in  the 
stage  of  red  hepatisation  is  not  autolysed,  in  the  grey  hepatised 
lung  autolysis  results  in  the  formation  of  lysine,  histidine,  leucine, 
and  tyrosine.3  The  changes  in  the  liver  in  phosphorus  poisoning 
are  due  to  autolytic  activity  getting  out  of  control,  for  Jacoby 
found  that  the  autolytic  changes  are  much  more  rapid  in  the 
excised  liver  of  animals  poisoned  with  phosphorus  than  in 
that  of  normal  animals ;  in  the  former  case  the  tissue  is 
reduced  to  a  fluid  mass  in  twelve  hours.4  Autolysis  may  be 
supposed  to  account  for  the  liquefaction  of  pus,  the  softening 
and  breaking  down  of  new  growths,  and  the  absorption  of 

1  Leathes,//.  of  Phys.  28,  360,  1902  ;  Dakin,  ib.  30,  84,  1903  ;  Cathcart, 
ib.  32,  299,  1905. 

2  Matthes,  S.  A.  51,  442,  1904. 

3  Simon,  D.  A.f.  k.  M.  70,  604,  1901. 

4  Jacoby,  H.-S.  Z.  30,  176,  1900. 


vii.]  AUTOLYSIS  IN  METABOLISM  147 

infarcts  and  thrombosed  parts.  The  absorption  of  gummata 
under  treatment  with  potassium  iodide  has  been  ascribed  to 
the  promotion  of  autolysis  by  this  drug.1  In  all  these  cases, 
however,  even  though  it  be  granted  that  the  operation  of  the 
cellular  enzymes  within  the  body  is  the  cause  of  the  change,  it 
may  be  said  that  the  cells  are  dead,  and  it  is  because  they  are 
dead  that  the  autolytic  process  is  manifested ;  the  fact  that  the 
process  is  in  these  conditions  found  at  work  within  the  body 
does  not  prove  that  they  play  a  part  in  the  metabolism  of  the 
normal  cells.  That  is  clearly  true  ;  but  normal  metabolism  is 
a  struggle  between  life  and  death,  in  which  life  just  manages 
to  get  the  upper  hand,  the  catabolic  processes  of  death  being 
more  than  compensated  for  by  the  restitutive  processes  of  life. 
We  have  to  choose  between  two  explanations :  either,  when 
cells  die,  enzymes  are  called  into  existence  to  act  as  licensed 
scavengers ;  or,  the  death  of  the  cells  manifests  itself  in  a 
disturbance  of  the  normal  balance  between  competing  changes, 
and  in  the  failure  of  those  conditions  that  with  a  normal 
circulation,  or  in  the  absence  of  poisons  such  as  those  intro- 
duced on  the  administration  of  phosphorus,  hold  in  check  and 
regulate  these  catabolic  processes  which  we  call  autolytic.  And 
if  we  choose  the  latter,  then  we  are  at  the  same  time  able  to 
understand  how  the  proteids  are  broken  down  in  normal 
metabolism,  and  how  it  is  that  the  muscles,  liver,  spleen,  and 
other  organs  are  in  starvation  able  to  supply  the  heart  and 
respiratory  muscles  with  all  the  nitrogenous  material  they 
require.  There  is  no  difficulty  in  suggesting  provisional  ex- 
planations of  how  it  is  that  the  action  of  these  cellular  enzymes 
during  normal  life  is  not  conspicuous,  as  it  is  in  the  pathological 
conditions  referred  to  above :  how  it  is  that  we  do  not  find  the 
products  of  their  action  normally  in  the  liver,  for  instance,  as 
we  do  after  poisoning  with  phosphorus ;  it  may  be  that  these 
products  undergo  further  changes  on  the  spot,  or  that  they  are 
removed  by  the  circulation  to  be  disposed  of  elsewhere  :  how  it 
is,  again,  that  the  organs  do  not  normally  liquefy  and  disappear 
under  their  action ;  it  may  be  that  this  is  held  in  check  by 
1  Oswald,  B.  Cbl.)  iii.,  367,  1905. 


148  PROTEID  CATABOLISM  [LECT. 

anti-catalytic  agents — Hedin  showed  that  the  serum  of  the  ox 
contains  such  a  substance  for  the  cellular  enzyme  of  the  spleen 
and  other  organs l — or  it  may  be  that  it  is  compensated  for  by 
the  due  adjustment  of  restitution  processes,  the  conditions  obtain- 
ing for  a  local  reversion  of  the  process. 

However  this  may  be,  even  before  the  cellular  proteolytic 
enzymes  came  to  light,  the  catabolism  of  proteids  was  very 
commonly  conceived  of  as  being  in  the  first  instance  simply 
hydrolytic.  It  has  been  obvious  for  many  years  that  such  a 
conception  was  compatible  with  known  facts  :  leucine,  tyrosine, 
aspartic  acid,  and  glycocoll,  when  taken  with  food  are  not 
excreted  ;  their  nitrogen  is  found  in  the  urine  as  urea,  and  the 
carbon,  so  far  as  is  known,  undergoes  the  same  fate  as  that  of 
the  proteids  themselves  :  no  unaltered  glycocoll,  alanine,  leucine, 
or  phenyl-alanine  is  found  in  the  urine,  even  with  the  delicate 
reagent  for  such  substances,  naphthalene  sulphonyl  chloride, 
after  administering  3  to  8  grammes  of  them  to  rabbits.2 

That  a  hydrolytic  resolution  of  proteids  does  form  part  of 
normal  catabolic  processes,  is  indicated  by  the  fact  that  some  of 
the  products  of  this  change  are  found  in  the  bile  and  urine, 
fixed  as  it  were  by  condensation  with  other  compounds,  and  so 
escaping  further  destructive  changes,  in  much  the  same  way  as 
glycuronic  acid  does.  Glycocoll  is  thus  fixed  by  condensation 
with  both  cholalic  and  benzoic  acids ;  diamido-valerianic  acid  or 
ornithine,  which  is  formed  by  the  hydrolysis  of  arginine,  is  in 
birds  similarly  combined  with  benzoic  acid ;  and  cystine,  after 
oxidation  of  the  mercaptan  group  and  the  loss  of  carbonic  acid, 
is,  it  is  also  known,  the  source  from  which  the  taurine  in 
taurocholic  acid  is  derived.3  It  is  difficult  to  account  for  the 
occurrence  of  these  substances  in  the  organism  otherwise  than 
by  supposing  them  to  be  formed  by  the  hydrolysis  of  proteid, 
and  if  so,  it  is  difficult  to  suppose  that  they  are  the  only  parts 
of  the  proteid  molecules  thus  split  off,  or  that  hydrolysis  in  the 
ceils  is  in  its  broad  outlines  different  from  hydrolysis  effected 

1  Hedin,//.  of  Phys.  30,  195,  1903. 

2  Abderhalden  and  Bergell,  H.-S.  Z.  39,  9,  1903. 

3  v.  Bergmann,  H.  B.  4,  132,  1903. 


vii.]  AMIDO  ACIDS  FOUND  IN  METABOLISM  149 

by  the  ferments  secreted  in  digestion,  or  by  those  that  act  in 
autolysis  outside  the  body. 

The  isolation  of  the  products  of  this  action  from  the  organs 
of  the  body  after  death,  except  in  cases  in  which  the  risk  of 
post-mortem  autolytic  changes  giving  rise  to  them  was  actually 
foreseen  and  avoided,  is  not  a  secure  foundation  for  an  argument 
on  this  point.  And  more  striking  than  the  fact  that  arginine 
has  been  isolated  from  the  spleen,  cystine  from  the  liver,  leucine 
from  most  of  the  organs  of  the  body,  and  so  forth,  is  the  fact 
that  from  the  normal  liver  and  intestine,  examined  with  due 
precautions  against  this  risk,  it  has  not  been  possible  to  isolate 
those  even  that  are  most  easily  isolated,  such  as  leucine  and 
tyrosine.  And  the  liver  and  intestine  we  know  contain,  at  any 
rate  after  removal  from  the  body,  the  necessary  enzymes  for 
their  formation.  But  if  we  cannot  base  our  belief  that  the 
first  step  in  proteid  catabolism  in  the  cells  is  hydrolysis  upon 
the  presence  of  the  resulting  substances  in  the  tissues,  their 
absence  need  not  cause  us  to  abandon  it.  We  are  apt  to 
form  an  exaggerated  notion  of  the  rate  at  which  proteid 
catabolism  is  carried  on  in  the  body.  Even  supposing  that 
the  urea  in  the  urine  is  a  measure  of  proteid  catabolism  in  the 
tissues  (which  it  is  probably  wrong  to  do),  a  man  of  70  kg.  in 
nitrogenous  equilibrium  on  120  g.  of  proteid  daily,  uses  up 
5  g.  of  proteid  on  an  average  in  an  hour  in  his  whole  body. 
The  amount  of  proteid  in  his  body,  disregarding  his  blood, 
must  be  considerably  more  than  5  kg.,  for  the  muscles  alone 
form  40  per  cent,  of  his  weight,  and  since  they  contain  about 
20  per  cent,  of  proteid,  they  alone  account  for  more  than  this. 
The  average  rate  at  which  proteid  is  used  up  is  therefore  con- 
siderably less  than  o.i  per  cent,  per  hour:  this  means  that  100  g. 
of  tissue,  containing  20  per  cent,  of  proteid,  will  use  up  on  an 
average  less  than  20  mg.  of  proteid  in  an  hour,  and  produce 
in  that  time  less  than  4  mg.  of,  say,  leucine,  forming  one-fifth  of 
the  weight  of  the  proteid.  So  that,  in  order  for  the  products 
of  proteid  hydrolysis  to  occur  in  any  organ  in  sufficient  quantity 
to  be  isolated  in  an  ordinary  experiment,  it  would  be  necessary 
to  suppose  either  a  rate  of  formation  very  considerably  greater 


150  PROTEID  CATABOLISM  [LECT. 

than  the  average  rate,  or  else  stagnation  of  the  other  processes 
by  which  these  products  are  further  acted  upon  or  removed. 

We  are  also  apt  to  form  our  notion  of  the  stability  of 
substances  that  occur  in  the  body  from  their  behaviour  when 
isolated  and  examined  outside  the  body.  Polysaccharides,  fats, 
and  proteids  alike,  when  isolated,  may  require  energetic  treat- 
ment in  order  to  separate  them  into  their  component  parts. 
But  in  living  organisms  the  work  of  hydrolysis  and  condensation 
is  so  simply  effected,  and  with  so  little  if  any  loss  of  energy, 
that  we  ought  to  accustom  ourselves  to  quite  different  notions 
of  their  stability  in  physiology.  To  take  the  most  familiar 
instances :  sugar  is  built  up  in  the  liver  into  glycogen  at  one 
moment,  to  be  reduced,  as  we  believe,  to  its  former  condition 
as  sugar  again  the  next,  and  again  may  become  glycogen  a 
moment  later  on  reaching  the  cells  in  other  parts.  The  fatty 
acids  are  divorced  from  glycerine  to  enter  the  intestinal 
epithelium,  and  reunited  as  they  leave  it  to  enter  the  lymph, 
and  it  seems  probable  that  they  are  again  separated  on  reaching 
the  blood,  and  remain  separated  till  they  settle  down  in  the 
connective  tissues.  Even  there,  too,  we  find  that  agencies  exist 
for  bringing  about  another  separation,  which  probably  takes 
place  before  the  next  stage  in  the  life  history  of  these  groups 
is  entered  upon.  It  is  only  putting  our  physiological  concep- 
tions of  the  proteids  in  the  body  on  a  footing  with  those  that 
we  have  to  form  of  the  fats  and  carbohydrates,  if  we  regard 
them  as  labile  aggregations  of  amido  acids  which  in  the  body 
have  far  less  cohesion  than  their  behaviour  when  isolated 
suggests. 

There  is  therefore  much  that  points  to  the  hypothesis, 
which  is  in  fact  commonly  adopted,  that  proteid  catabolism 
begins  by  a  resolution  of  the  proteids  into  their  simple  com- 
ponent parts,  such  as  is  effected  also  in  digestion.  If,  as  is 
probable,  this  is  a  change  which,  like  the  corresponding  change 
in  fats  and  carbohydrates,  is  practically  isothermic,  then  this 
change  is  merely  a  preamble  to  those  in  which  the  proteids 
are  made  use  of  as  a  source  of  energy  ;  and  proteid  catabolism, 
in  so  far  as  this  is  concerned  with  heat-production  or  work 


vii.]  DENITRIFICATION  OF  AMIDO  ACIDS  151 

done  in  muscles  or  elsewhere,  is  the  catabolism  of  nothing 
more  complex  than  the  amido  acids,  simple  and  compound, 
thus  set  free. 

The  accounts  of  the  course  of  proteid  metabolism,  starting 
from  the  proteids  themselves,  which  it  has  hitherto  been  possible 
to  give,  have  at  this  point  had  to  cut  matters  short  and  jump 
to  the  end-products.  But  it  is  now  possible  that  a  further  step 
may  be  taken.  It  has  recently  been  shown  that  in  many  of 
the  organs  and  tissues  a  reaction  takes  place,  the  work  pre- 
sumably of  an  enzyme,  which  shows  itself  by  the  liberation  of 
ammonia  from  amido  acids.1  Lang  in  Hofmeister's  laboratory 
treated  the  pulp  obtained  from  various  organs  very  thoroughly 
with  toluene,  by  shaking  mechanically  for  some  time,  and  then 
estimated,  after  keeping  them  for  some  hours  at  the  body- 
temperature  in  an  incubator,  the  amount  of  ammonia  present 
in  samples  to  which  various  amido  acids  had  been  added,  and 
in  others  to  which  no  addition  had  been  made.  More  ammonia 
was  found  when  leucine  or  glycocoll,  tyrosine  or  cystine,  was 
added,  in  almost  all  of  a  large  number  of  experiments.  The 
most  marked  action  was  with  the  intestine,  liver,  or  pancreas 
treated  with  glycocoll  or  leucine.  But  it  was  clear  that  the 
toluene,  as  is  unfortunately  so  commonly  the  case  with  anti- 
septics, exerted  a  very  unfavourable  influence  on  the  reaction ; 
since  liver  pulp,  prepared  as  far  as  possible  aseptically,  produced 
more  ammonia  from  glycocoll  in  an  hour  and  a  half  than  was 
produced  by  the  liver  in  the  presence  of  toluene  in  any  other 
experiment  after  several  days.  Sufficient  growth  of  the  few 
imported  bacteria  can  hardly  have  taken  place  in  this  short 
time  to  have  produced  any  detectable  ammonia. 

These  results  are  an  extension  of  results  arrived  at  pre- 
viously by  Jacoby,2  who  found  that,  in  the  fluid  obtained  from 
the  liver  after  grinding  it  up  with  sand,  the  ammonia  increased, 
and  that  this  ammonia  was  derived  from  substances  which  like 
the  amido  acids  do  not  give  up  their  nitrogen  when  boiled  with 
acids.  O.  Loewi  had  also  shown  that  the  amido  group  in 

1  Lang,  H.  B.,  v.,  321,  1904. 

2  Jacoby,  H.-S.  Z.  30,  149,  1900. 


152  PROTEID  CATABOLISM  [LECT. 

glycocoll  was  changed,  in  the  presence  of  a  pulp  of  liver  cells, 
into  some  substance  soluble  in  alcohol,  not  urea,  which  like 
urea  gives  up  ammonia  when  treated  with  fixed  alkalies,  the 
firm  union  of  nitrogen  to  carbon  as  it  exists  in  the  amido  acid 
having  been  dissolved.  It  seems,  therefore,  that  the  power  of 
removing  ammonia  from  amido  acids  may  be  a  general  property 
of  the  cells  of  many  tissues  in  the  body  ;  just  as  we  know  that 
bacteria  remove  ammonia  from  tyrosine  in  putrefaction,  or  from 
tryptophane,  and  from  urea.  And,  at  any  rate,  this  property  is 
marked  in  the  intestinal  mucous  membrane  and  liver.  This 
explains  the  fact  observed  by  Nencki l  and  his  fellow-workers, 
that  the  amount  of  ammonia  in  the  portal  blood  is  during 
digestion  greater  than  that  in  the  systemic  blood,  as  much  as 
four  times  the  amount  being  sometimes  found :  ammonia  is 
split  off  from  amido  acids  in  the  intestine,  conveyed  to  the  liver, 
and  there  removed  from  the  blood  and  converted  into  urea. 
The  stomach,  intestines,  liver,  and  pancreas  all  contain,  accord- 
ing to  the  same  observers,  considerably  more  ammonia  than  the 
muscles,  brain,  kidneys,  or  spleen. 

Lang's2  experiments,  it  may  be,  require  extension  and 
confirmation  under  varying  conditions,  though  they  leave  no 
doubt  about  the  main  fact.  According  to  them,  however,  while 
some  of  the  amido  acids,  unlike  leucine  and  glycocoll,  are 
apparently  hardly  acted  on  at  all,  phenyl-alanine,  for  instance, 
and  in  some  cases  cystine  and  tyrosine,  amides  such  as 
acetamide,  but  in  a  much  more  marked  degree  the  amides  of 
amido  acids,  asparagine,  and  glutamine,  give  up  ammonia  as 
well.  Now  it  is  known  that  a  certain  amount  of  the  nitrogen  in 
proteids  is  given  off  as  ammonia  in  hydrolysis  both  by  enzymes 
and  mineral  acids,  and  this  nitrogen  is,  therefore,  commonly 
referred  to  as  the  "amide"  nitrogen  of  the  proteid  molecule. 
This  amide  nitrogen  may,  therefore,  be  fairly  assumed  to 
contribute  to  the  nitrogen  removed  from  proteids  in  the  liver 
as  well  as  other  organs,  whether  the  removal  be  effected  in  the 

1  Nencki  and  Zaleski,  S.  A.  36,  385  ;  and,  A.  St  P.,  1895.     Salaskin  and 
Zaleski,  ff.-S.  Z.  246 ;  cf.  Folin,  H.-S.  Z.  37,  174,  1902. 

2  Lang,  H.  B.  5,  340,  1904. 


vii.]  LIBERATION  OF  AMMONIA  153 

course  of  proteid  hydrolysis  or  in  this  special  denitrifying 
process.  The  amide  nitrogen  constitutes  in  the  case  of  casein 
as  much  as  1 3  per  cent  of  the  whole  nitrogen  ;  in  other  proteids, 
serum-  and  egg-albumin  and  the  primary  albumoses  obtained 
from  fibrin,  generally  about  7  or  8  per  cent. ;  while  in  gelatine 
it  is  less  than  2  per  cent.1  The  denitrification  of  amides  is, 
however,  not  quite  a  universal  reaction,  to  judge  from  the  fact 
that  oxamic  acid  is  not  converted  into  oxalic  acid  at  all,  but 
appears  to  be  converted  directly  into  urea  when  administered  to 
rabbits.  This  is,  however,  rather  an  exceptional  form  of  amide.2 

Whether  the  diamido  acids  contribute  to  this  ammonia 
formation,  we  do  not  know :  but  from  the  following  fact  it  seems 
probable.  Just  as  Neuberg  and  Langstein  found  that  after 
administration  of  alanine  to  rabbits  in  considerable  doses,  small 
quantities  of  the  denitrified  product,  lactic  acid,  escaped  oxida- 
tion and  appeared  in  the  urine,3  so  P.  Mayer  showed  that 
diamido-propionic  acid  injected  subcutaneously  into  rabbits 
gave  rise  to  the  excretion  of  a  small  quantity  of  the  corre- 
sponding dioxy-acid,  glyceric  acid ;  the  small  yield  not 
necessarily  meaning,  of  course,  that  no  more  was  formed,  since 
such  a  substance  as  glyceric  acid  could  hardly  be  expected  to 
run  the  gauntlet  in  the  body  and  appear  to  any  great  extent 
in  the  urine  unaltered.4  Lysine,  therefore,  and  the  ornithine 
set  free  by  arginase  in  the  body,5  may  very  probably  share  the 
fate  of  the  mono-amido  acids  and  lose  their  nitrogen  before  they 
are  otherwise  acted  on  in  the  cells. 

Now  the  amido  acids  are  nitrogenous  substances  in  which 
the  combination  of  carbon  and  nitrogen  is  particularly  firm,  as 
is  obvious  from  the  stability  of  these  compounds  under  the 
action  of  the  proteoly tic  enzymes  and  of  boiling  mineral  acids. 
The  removal  of  the  nitrogen  from  amido  acids  by  the  cells  of 
the  body  is  a  remarkable  reaction,  and  one  that  has  hitherto 

1  Hausmann,  H.-S.  Z.  27,  95,  and  29,  136,  1899  and  1900. 

2  Schwarz,  S.  A.  41,  60,  1898. 

3  Neuberg  and  Langstein,  M.J.^  p.  603,  1903. 

4  P.  Mayer,  H.-S.  Z.  42,  59,  1904. 

5  Kossel  and  Dakin,  H.-S.  Z.  41,  321,  1904. 


154 


PROTEID  CATABOLISM 


[LECT. 


not  been  generally  taken  into  account  in  considering  the  course 
and  nature  of  proteid  catabolism.  We  do  not  know  to  what 
extent  it  takes  place  in  either  the  assimilation  of  proteid  food 
or  cell  metabolism.  But  in  future  account  must  be  taken  of  it, 
and  a  little  reflection  must  show  that  it  may  have  important 
bearings  on  some  of  the  most  prominent  problems  of  nitrogenous 
metabolism. 

In  the  first  place  it  may  be  as  well  to  take  note  of  the  fact 
that  the  removal  of  the  nitrogen  does  not  very  materially  affect 
the  energy  value  of  the  acids.  The  heat  equivalents  of  some  of 
the  amido  acids  and  the  corresponding  fatty  and  oxy-acids  are 
given  in  the  following  table  : — 


Cal. 
per  1  g. 

Cal. 
per  1  gm.-mol. 

Difference 
per  cent. 

Leucine        '», 

G.$2 

854-9 

1 

Caproic  acid  . 

7.16 

830.2 

j           3 

Leucic  acid   .        .        . 

... 

... 

... 

Alanine          .'    ;    ^Wu 
Propionic  acid 

4.36 

4.95 

389 
366.9 

1  » 

Lactic  acid     . 

(?)  3.7 

(?)  338  1 

15 

33i 

... 

Glycocoll       .        .        . 
Acetic  acid     .         .         ,"" 

3.13 
3.49 

^35 
209 

10.4 

Glycollic  acid     .*  ".  "      . 

2.10 

160 

22 

1  This  has  not  been  directly  determined  apparently ;  but,  as  was  pointed  out  in 
Lecture  III.,  p.  63,  has  probably  about  this  value. 

The  figures  given  in  the  last  column  show  the  difference  of 
the  energy  value  of  the  fatty  acid  as  compared  with  that  of  the 
corresponding  amido  acid,  reckoned  in  percentage,  of  the  latter. 
Even  in  the  case  of  the  smallest  molecule,  glycocoll,  it  amounts 
only  to  about  10  per  cent.  We  do  not  know  what  the  non- 


vn.]     NOT  TO  BE  MEASURED  BY  UREA  EXCRETION      155 

nitrogenous  compounds  left  after  removal  of  the  nitrogen  are : 
it  is  perhaps  more  likely  that  they  are  the  oxy-acids  than  the 
unoxidised  fatty  acids,  although  it  is  oxyphenyl  propionic  acid, 
not  oxyphenyl  lactic  acid,  that  is  produced  by  bacteria  from 
tyrosine ;  and  in  that  case  the  energy  set  free  will  be  a  larger 
proportion  of  the  whole.  But,  even  so,  if  proteids  in  assimila- 
tion are  first  hydrolysed,  and  then  to  any  considerable  extent 
the  amido  acids  denitrified,  the  resulting  products  would  hardly 
have  given  up  more  than  about  10  to  15  per  cent,  of  the 
energy  in  the  proteid  molecule  from  which  they  were 
derived. 

In  other  words,  the  nitrogen,  or  a  great  part  of  it,  may  be 
removed  from  the  proteid,  converted  into  urea,  and  expelled 
with  the  urine  before  the  oxidation  of  the  rest  of  the  molecule 
has  been  started  upon  ;  and  the  fact  that  we  can  trace  in  the 
urine  excreted  in  a  given  time,  all  or  the  greater  part  of  the 
nitrogen  of  the  proteid  taken  at  a  meal,  tells  us  nothing  what- 
ever about  the  fate  of  that  part  of  the  proteid  which  contains,  it 
may  be,  as  much  as  80  or  90  per  cent,  of  the  total  energy  of  the 
proteid  food.  Proteid  metabolism  in  so  far  as  it  is  concerned 
with  the  evolution  of  energy,  proteid  metabolism  in  its  exo- 
thermic stages,  may  be  almost  entirely  non-nitrogenous  meta- 
bolism. Our  habit  of  looking  upon  the  appearance  of  so  much 
urea  in  the  excreta  as  a  sign  that  so  much  proteid  has  been 
"  used  up,"  of  calculating  the  total  energy  corresponding  to  that 
amount  of  proteid,  and  saying  that  that  amount  has  been 
derived  from  the  combustion  of  proteid,  may  be  based  entirely 
upon  a  misconception.  We  shall  have  in  future  to  bear  this 
reaction  in  mind  :  it  has  been  proved  in  the  case  of  the  cells 
which  are  the  first  cells  in  the  body  with  which  the  absorbed 
nitrogenous  substances  come  into  relations ;  it  is  a  reaction 
which  leaves  the  greater  part  of  the  energy  of  the  proteid 
molecule  untouched,  but,  nevertheless,  puts  the  greater  part  of 
the  nitrogen  in  the  way  for  being  thrown  off  as  urea  in  the 
urine.  It  is  well  known  that  in  dogs,  as  in  man,  after  a  proteid 
meal,  the  urea  excretion  rises,  and  in  the  first  six  or  seven 
hours  this  rise  may  account  for  fully  half  the  nitrogen  of  the 


156  PROTEID  CATABOLISM  [LECT. 

meal,  and  in  a  few  hours  more  for  almost  the  whole  of  it ; l  and 
since  in  dogs,  after  a  large  meal  of  proteid,  absorption  from  the 
intestine  is  known  to  be  extended  often  over  fourteen  hours  or 
more,  the  whole  of  the  nitrogen  may  be  traced  into  the  urine 
within  an  hour  or  two  of  its  absorption.  It  is  true  that  the 
carbonic  acid  excretion  is  simultaneously  somewhat  increased, 
but  this  is  to  be  explained  almost  entirely,  in  the  first  place,  by 
the  increased  muscular  activity  of  the  digestive  tract  which,  it  is 
known,  influences  the  carbonic  acid  output  just  as  the  activity 
of  the  voluntary  muscles  does  ;  and  in  the  second  place,  by  the 
increased  activity  of  the  digestive  and  excretory  organs :  but 
there  is  no  strict  parallelism  between  the  excretion  of  urea  and 
carbonic  acid.  The  two  main  end-products  of  proteid  meta- 
bolism, urea  and  carbonic  acid,  are,  to  a  great  extent,  produced 
independently  of  each  other,  and  the  reactions  which  result  in 
the  discharge  of  the  nitrogen  are  not  those  in  which  energy  is 
set  free,  work  done,  and  carbonic  acid  produced.  This  rise  in 
the  rate  of  excretion  of  urea  after  a  proteid  meal,  which  we 
have  learned  to  regard,  with  both  Voit  and  Pfliiger,  as  a  sign 
that  the  cells  of  the  body  prefer  to  use  proteids  for  all  their 
requirements,  if  only  they  can  get  them,  and  that  it  is  only 
when  the  proteids  are  used  up  that  the  cells  fall  back  upon  the 
non-nitrogenous  stores,  we  must  now  be  prepared  to  learn  is  a 
sign  of  nothing  of  the  kind,  but  rather  a  sign  that  the  body  has 
no  need  for  all  this  nitrogen,  and  that  it  must  be  got  rid  of 
before  the  really  valuable  part  of  the  proteid  molecules  is 
admitted  into  the  general  circulation. 

One  of  the  most  familiar  facts  with  regard  to  nitrogenous 
metabolism  is,  that  the  amount  of  nitrogen  excreted  in  starvation 
is  much  less  than  the  minimum  amount  that  must  be  given  in 
the  form  of  proteid  in  order  to  maintain  nitrogenous  equilibrium. 
This  fact  appears  in  a  new  light,  if  we  suppose  that  a  long 
succession  of  generations  in  the  past,  which  have  lived  from 
choice  or  necessity  on  a  diet  rich  in  proteids,  have  handed  down 
to  us,  as  our  inheritance,  a  constitution  in  which  arrangements 
exist  for  the  removal  of  nitrogen  from  a  considerable  part  of 

/.  7.247,  1876. 


vii.]  NITROGEN  CANNOT  BE  STORED  157 

this  proteid.  The  fact  that  the  amount  of  proteid  taken  is 
readjusted  to  suit  the  actual  needs  of  the  body,  though  it  makes 
these  arrangements  unnecessary,  will  not  necessarily  remove 
them.  The  denitrifying  enzyme,  which  has  been  trained  to 
keep  guard  over  the  entrances  by  which  nitrogenous  substances 
are  admitted  into  the  body,  will  continue  to  levy  its  toll  of 
nitrogen,  even  when  the  amount  of  proteid  presented  to  it  is  no 
more  than  the  tissues  which  it  serves  actually  require. 

Again,  it  is  one  of  the  cardinal  laws  of  proteid  metabolism 
that  the  store  of  nitrogenous  substances  in  the  body  is  not 
increased  by,  or  not  in  proportion  to,  an  increase  in  the  nitrogen 
intake.  There  is  a  classical  experiment  of  Voit's  which  is 
everywhere  referred  to,  in  which  a  dog,  after  being  kept  in 
nitrogenous  equilibrium  on  a  daily  ration  of  500  g.  of  meat,  was 
given  three  times  that  amount  on  seven  days  in  succession. 
On  the  very  first  day  the  nitrogen  excreted  corresponded  to 
1222  g.  of  meat,  on  the  third  to  1390  g.,  and  on  the  seventh 
nitrogenous  equilibrium  was  re-established.  In  seven  days 
7  kg.  of  meat  had  been  given  above  and  beyond  what  was 
sufficient  for  the  animal's  normal  existence ;  yet  only  about 
1 1  g.  of  the  nitrogen  contained  in  this  meat  had  been  retained 
in  the  body,  and  when  the  daily  allowance  was  reduced  to 
1000  g.  of  meat,  or  double  the  amount  that  was  formerly 
sufficient,  most  of  what  had  been  gained  was  lost  in  the  next 
five  days.  Siven,  in  his  experiments  on  himself,1  came  across 
the  same  phenomenon.  With  an  exceedingly  low  nitrogen 
intake,  maintained  for  some  weeks,  he  lost  about  38  g.  of 
nitrogen.  Then,  during  a  week,  with  12  g.  daily,  he  gained 
only  14.5  g.  in  all,  and  in  the  succeeding  six  days,  with  a  daily 
intake  of  22.6  g.,  a  further  6.5  g.,  making  a  gain  altogether  of 
only  21  g.  in  thirteen  days.  He  concluded,  as  has  been 
commonly  done  in  such  circumstances,  that  it  is  a  tendency  of 
the  body,  even  with  an  abundant  supply  of  nitrogen,  and  after 
a  period  of  nitrogen  loss,  to  strive  towards  nitrogen  equilibrium  ; 
and  commenting  on  this,  lays  it  to  the  credit  of  the  "  circulating 
proteid  "  theory  of  Voit.  This  phenomenon  has  always  appeared 
1  Siven,  Sk.  A.  u,  308,  1901. 


158  PROTEID  CATABOLISM  [LECT. 

a  most  remarkable  one,  and  if  we  may  explain  it  in  the  light  of 
what  we  have  recently  learnt  of  the  reactions  carried  out  in  the 
cells  of  the  intestine  and  liver,  it  may  be  a  relief  to  many  who 
have  felt  the  strain  of  adhesion  to  the  current  explanations. 
We  may  then  see  that  much  of  the  proteid  does  not  get  past  the 
liver  as  nitrogenous  matter  at  all ;  all  excess  of  proteid  above 
what  is  indispensable  for  repair  is  stripped  of  its  nitogen  and 
can  only  reach  the  tissues  in  combinations  in  which  nitrogen 
does  not  occur ;  the  nitrogen  that  is  not  wanted  is  removed  as 
ammonia,  converted  into  urea,  and  expelled  from  the  system 
by  the  kidneys.  And  when  nitrogen  is  wanted  to  repair  loss, 
even  then,  because  of  this  tendency  of  the  substances  derived 
from  proteid  food  to  lose  their  nitrogen  before  admission  to  the 
body,  only  a  small  portion  of  them  can  run  through  past  the 
liver  and  become  available  for  this  repair.  The  question 
whether  the  urea  is  formed  from  "  circulating "  or  "  tissue- 
proteid  "  does  not  come  in  at  all,  and  we  can  escape,  it  may  be, 
from  the  dilemma  of  having  to  take  for  our  own,  one  or  other 
of  those  two  historical  watchwords,  each  of  which  has  been  so 
stoutly  defended  in  the  literature  of  physiology. 

The  ideas  underlying  this  famous  controversy  are  doubtless 
fundamental,  and  must  be  extended  to  include,  or,  rather  perhaps 
confined  to,  the  non-nitrogenous  components  of  cells.  What  is 
the  relation  of  cell-protoplasm  and  of  nucleo-plasm  to  the  non- 
nitrogenous  material  in  the  oxidation  of  which  the  vital 
transformations  of  chemical  energy  are  made  manifest  ?  Does 
it  become  a  part  of  that  protoplasm,  or  is  it  acted  on  by  mere 
contact  with  it  ?  It  may  be  that  in  some  such  form  as  this 
we  may  look  to  this  question  reappearing  in  the  future.  But 
it  is  of  little  use  trying  to  formulate  it  now,  since  for  the 
present  we  hardly  have  sufficient  data  to  enable  us  to  ask 
ourselves  intelligent  questions  on  such  subjects.1 

1  O.  Folin  has  recently  called  attention  to  the  weakness  of  the  principal 
experiment,  which  has  very  commonly  been  regarded  as  giving  the 
supporters  of  the  organ  proteid  theory  the  best  of  the  argument :  Schondorff  s 
well-nourished  dog,  before  the  perfusion  experiment,  was  turning  out 
nitrogen  at  the  rate  of  38  g.  a  day,  or  nearly  1.5  g.  an  hour  :  the  blood 


vii.]  THE  NITROGEN  MINIMUM  159 

But  whatever  significance  we  read  into  the  work  that  has 
come  from  Hofmeister's  laboratory,  the  fact  of  course  remains 
that  animals  die  without  proteid,  or,  at  any  rate,  without  the 
products  of  proteid  hydrolysis  in  their  food.  And  therefore  it 
is  hardly  to  be  supposed  that  the  nitrogen  of  the  whole  of 
these  substances  is  removed  before  they  can  get  past  the  liver, 
so  that  for  replacing  tissue  the  combinations  have  to  be  re- 
synthesised.  If  that  were  so,  it  should  be  possible  to  maintain 
animal  life  on  ammonia  and  non-nitrogenous  foods.  The 
utmost  that  the  data  warrant  is  that  from  a  part  of  them  the 
nitrogen  is  so  removed,  it  may  be  a  considerable  part,  and 
sufficient  to  account  for  the  phenomena  that  we  have  just  been 
discussing.  But  some  of  it  must  remain. 

What  is  suggested  before  all  things  by  these  results,  as  it 
appears,  is  that  it  is  no  longer  possible  to  take  the  rate  of  urea 
formation  as  necessarily  a  measure  of  proteid  metabolism, 
whether  by  that  we  mean  the  rate  at  which  cell  protoplasm 
decays  or  merely  the  rate  at  which  proteid  is  used  as  a  source 
of  energy  in  the  body.  It  is  not  a  measure  of  the  true  proteid 
catabolism,  the  decay  of  living  nitrogenous  matter,  because  a 
great  part  of  it  is  formed  from  nitrogen  that  has  never  been 
beyond  the  liver,  and  it  is  not  a  measure  of  the  energy  derived 
from  proteid,  because  it  is  largely  derived  from  proteid  by 
reactions  which  leave  the  energy  value  of  the  molecules  from 
which  it  is  derived  but  little  altered. 

Further,  it  must  occur  to  us  that  if  a  considerable  part  of 
the  proteid  is  only  allowed  to  reach  the  tissues  of  the  body 
generally  in  a  non-nitrogenous  form,  it  should  be  possible  to 
replace  this  part  by  non-nitrogenous  food-stuffs  from  the  outset ; 
and  that  the  low  nitrogen  dietaries  that  in  some  races  are 
found  to  be  compatible  with  great  physical  activity  may  be 

perfused  for  four  and  a  half  hours  through  the  hind-limbs  and  liver  collected 
25  mg.  of  nitrogen  in  the  form  of  urea.  In  the  entire  living  animal  during 
that  time  it  would  have  conveyed  about  6  g.  of  nitrogen  as  urea  to  the 
kidney,  or  considerably  more  than  two  hundred  times  as  much.  As  Folin 
points  out,  25  mg.  of  nitrogen  is  not  sufficient  foundation  for  so  weighty  a 
superstructure. 


160  PROTEID  CATABOLISM  [LECT. 

explained  by  taking  this  into  account.  In  point  of  fact,  this  is 
just  the  conclusion  that  certain  workers  on  proteid  metabolism 
in  laboratories  both  in  Europe  and  America  have  arrived  at, 
quite  apart  from  these  considerations. 

Siven1  found  in  experiments  on  himself  that  he  could  lead 
his  ordinary  life  and  maintain  nitrogenous  equilibrium  without 
losing  weight,  when  taking  only  between  4  and  5  g.  of  nitrogen 
daily,  provided,  of  course,  that  the  total  Calories  supplied  in  his 
food  amounted  to  a  little  over  40  per  kg. 

Landergren,2  in  four  experiments  on  a  man  of  70  kg.,  found 
that  if  carbohydrates  were  given  in  sufficient  quantity  to  provide 
from  38  to  45  Calories  per  kg.,  and  no  nitrogen,  except  about 
i  g.  daily,  which  was  inseparable  from  the  forms  of  carbo- 
hydrate food  employed,  then  the  nitrogen  excretion  sank  on 
the  fourth  day  to  from  3  to  4  g. ;  about  a  fifth,  that  is,  of  the 
amount  ordinarily  excreted. 

Chittenden  finds  that  he  himself  and  his  collaborators,  and 
also  men  undergoing  military  training,  and  students  actively 
engaged  in  athletic  competitions,  maintain  their  weight  and 
nitrogenous  equilibrium  on  a  diet  containing  only  from  30  to  50 
per  cent,  of  the  amount  of  proteid  which  has  been  commonly 
regarded  as  normal  for  man  ;  and  in  some  cases  his  observations 
extend  continuously  over  several  months.3 

It  may  be,  therefore,  that  the  conventional  dietary  of  100  g. 
of  proteid,  or  more,  should  be  very  considerably  cut  down,  and 
that  that  part  of  this  allowance  of  proteid  which  is  merely 
denitrified  and  used  by  the  body  in  non-nitrogenous  forms  should 
be  habitually  replaced  by  carbohydrates,  and  in  part  by  fat. 
But  it  does  not  necessarily  follow  that  this  is  so,  or  that  it  is 
unphysiological  for  man  to  take  more  than  the  minimum  amount 
of  nitrogen  necessary  for  equilibrium,  any  more  than  it  is 
unphysiological  to  take  any  food  which  yields  more  than  the 
minimum  amount  of  faecal  refuse.  The  experiments  on  the 
nitrogen  minimum,  establishing  an  amount  that  holds  for  a  few 

1  Siven,  Sk.  A.  10,  91,  and  n,  308,  1900-1. 

2  Landergren,  Sk.  A.  14,  112,  1902. 

3  Chittenden,  Physiological  Economy  in  Nutrition,  Heinemann,  1905. 


vii.]  iS  THE  MINIMUM  NORMAL?  l6l 

days  or  weeks,  do  not  necessarily  prove  that  it  holds  for  pro- 
longed or  for  lifelong  use.  The  food  of  an  infant  in  the  second 
half  of  the  first  year  is  commonly  and  normally  about  two  pints  of 
milk  ;  even  taking  this  to  contain  only  1.5  per  cent,  of  proteids, 
that  gives  about  17  g.  daily,  or  2  g.  per  kg.,  and  this  estimate, 
which  is  certainly  not  high,  is  more  than  the  conventional  adult 
diet  provides,  and  from  five  to  ten  times  as  much  as  the 
minimum.  It  is,  indeed,  a  well-known  fact  that  the  rate  of  urea 
excretion  in  infancy  is  higher  in  proportion  to  the  body-weight 
than  at  any  other  period  of  life.  If  ten  times  the  minimum  rate 
is  the  normal  diet  provided  by  Nature,  then,  even  after  making 
full  allowance  for  the  necessities  of  growth,  the  minimum  can 
hardly  be  normal  for  the  adult,  nor  the  amount  ordinarily  taken 
a  very  great  deviation  from  the  prescriptions  of  Nature.  Again, 
if  the  removal  of  nitrogen  from  certain  amido  acids  is  established 
as  a  fundamental  physiological  reaction,  it  is  not  proved  that  it 
applies  to  all  the  compounds  of  nitrogen  formed  from  proteids  : 
it  is  said,  for  instance,  that  tyrosine  and  phenyl-alanine,  unlike 
most  of  the  amido  acids,  when  injected  into  the  blood,  do  not 
increase  the  urea  excretion,1  and  in  Lang's  experiments  tyrosine 
reacted  feebly,  if  at  all,  and  phenyl-alanine  not  at  all :  there  are 
nitrogen  compounds  in  proteids  which  are  of  a  different  nature 
altogether ;  histidine,  the  indol  group  in  tryptophane,  and  perhaps 
pyrrholidine  may  be  included  with  these;  and  one  of  these, 
histidine,  there  are  reasons  for  thinking,  contains  a  nitrogenous 
group  which  is  related  to  one  of  those  present  in  nucleic  acids. 
We  know  nothing  of  the  fate  of  the  nitrogen  in  these  compounds, 
but  it  may  be  that  one  or  other  of  them  is  required  in  larger 
amounts  for  cell  repair,  and  that  it  is  only  the  ordinary  amido 
acids  that  are  not  required  in  the  amount  commonly  taken. 

There  still  remains  that  side  of  proteid  catabolism  which 
represents  the  decay  of  protoplasm.  This,  we  may  suppose,  is 
the  source  of  the  nitrogenous  excreta  when  all  superfluous 
proteid  is  excluded  from  the  food — when  all  the  proteid  that  is 
merely  denitrified  in  the  process  of  assimilation  is  dispensed 
with.  In  the  experiments  of  Siven  and  Landergren  this  was 

1  Stoltz,  H.  B.,  v.}  25,  1903. 

L 


162  PROTEID  CATABOLISM  [LECT. 

shown  to  yield  about  one-fifth  of  the  amount  of  nitrogen 
commonly  taken  as  normal.  It  becomes  a  point  of  great 
interest  to  know  in  what  form  this  nitrogen  is  excreted.  The 
denitrification  processes  result,  we  must  suppose,  in  the  discharge 
of  nitrogen,  principally  in  the  form  of  urea,  with  possibly  some 
ammonia,  and  even  some  uric  acid  ;  since,  according  to  Wiener,1 
uric  acid  can  be  synthesised  in  the  mammalian  organism,  as  in 
birds,  from  urea  and  an  organic  acid  free  from  nitrogen  contain- 
ing a  chain  of  three  carbons.  If  these  excretory  substances, 
resulting  from  the  denitrification  process,  are  subtracted  from 
the  urine,  then  the  distribution  of  the  nitrogen  over  the  several 
different  nitrogenous  substances  of  the  urine  may  be  much 
altered.  For  the  breakdown  of  cell  protoplasm,  including  the 
nucleoplasm,  may  give  rise  to  these  same  substances  in  other 
proportions,  or  even  to  other  substances  altogether.  If,  therefore, 
on  a  diet  containing  the  minimum  amount  of  proteid,  or  better 
still,  a  diet  free  from  proteid,  but  of  a  sufficient  energy 
equivalent,  it  is  found  that  the  distribution  of  nitrogen  is 
altered,  this  might  fairly  be  regarded  as  a  corroboration  of  the 
hypothesis  to  which  we  have  been  led.  As  a  matter  of  fact, 
Folin  2  has  found  just  such  a  difference,  and  on  this  ground  has 
been  led  to  take  a  general  view  of  proteid  metabolism  which 
agrees  almost  exactly  with  the  one  to  which  the  work  of  Lang 
and  others  in  Hofmeister's  laboratory  has  led  us.  The  principal 
points  which  he  has  determined  are,  that  under  these  conditions 
the  nitrogen  in  the  form  of  urea  sinks  from  87  per  cent,  of  the 
total  nitrogen  to  about  60  per  cent,  and  the  inorganic  sulphates 
at  the  same  time  from  90  to  60  per  cent,  of  the  total  sulphur ; 
the  kreatinine  and  neutral  sulphur  in  absolute  amount  remain 
almost  unchanged,  in  percentage  of  the  total  nitrogen  or  sulphur 
are  therefore  greatly  increased,  the  kreatinine  nitrogen  rising 
from  3.6  per  cent  to  17.4  per  cent,  and  the  neutral  sulphur  from 
5  per  cent  to  26  per  cent  The  other  forms  of  nitrogen  which 
he  determined,  ammonia  and  uric  acid,  as  well  as  those  forms 
which  he  did  not  determine  directly,  but  estimated  together  by 

1  Cf.  infra^  page  187. 

2  Folin,  Am.JL  Phys.  13,  66,  and  117,  1905. 


vii.]  AFTER  DISCOUNTING  DENITRIFICATION  163 

subtracting  from  the  total  nitrogen  the  sum  of  the  various  forms 
of  nitrogen  directly  determined — all  these  three,  as  well  as  the 
ethereal  sulphates,  were  diminished  in  absolute  amount,  but  in 
percentage  of  the  total  nitrogen  or  sulphur  were  increased.  The 
diets  he  employed  for  his*  comparison  were,  on  the  one  hand,  an 
egg  and  milk  diet,  containing  about  19  g.  of  nitrogen,  but  no 
kreatine  and  no  purine ;  and  on  the  other,  a  starch  and  cream 
diet,  containing  only  about  I  g.  of  nitrogen. 

These  results  are  of  great  interest  and  importance,  and  fully 
justify,  as  I  believe,  the  complete  reconsideration  of  the  position 
of  physiology  as  to  the  nature  and  course  of  proteid  metabolism 
which  he  has  advocated. 

Taken  in  connection  with  the  account  we  have  been  led  to 
above,  they  indicate  that  the  catabolism  of  the  tissues  accounts 
for  all  the  kreatinine  excreted  on  a  diet  free  from  meat,  and  all 
the  neutral  sulphur,  a  larger  proportion  of  the  uric  acid,  ammonia 
and  ether  sulphates,  and  a  smaller  proportion  of  the  urea  and 
inorganic  sulphates  than  the  denitrification  processes  ;  while  the 
fact  that  the  reduction  of  the  proteid  supply,  so  as  to  exclude 
those  changes  which  the  proteid  food  is  liable  to  in  the  digestive 
organs  and  liver,  is  accompanied  by  a  diminution  of  only  those 
nitrogenous  constituents  of  the  urine  which  we  know  may  be 
formed  from  ammonia,  is  some  confirmation  of  the  significance 
ascribed  above  to  the  denitrification  processes. 

The  idea  that  a  nitrogen-free  residue  might  result  at  some 
stage  of  proteid  metabolism  before  the  final  one,  is  by  no  means 
new.  Minkowski,  in  calculating  the  utmost  conceivable  amount 
of  sugar  that  could  be  formed  from  proteid,  claimed  the  whole  of 
the  carbon  that  was  not  wanted  for  urea ;  Seegen,  in  his  theory 
of  the  origin  of  the  sugar  in  the  liver  from  peptone,  and  many 
others,  have  more  or  less  dimly  tried  to  realise  some  such  stage 
in  the  metabolism  of  proteids,  as  Lang's  experiments  and  those 
that  preceded  them  have  revealed.  The  problem  of  the 
synthesis  of  sugar  from  proteid  has  taken  a  new  aspect,  and  one 
step  forward  has  been  taken  which  may  lead  to  others,  and 
finally  make  clear  how  the  change  may  be  effected.  As  we 
have  already  seen  in  an  earlier  lecture,  several  experiments  have 


164  PROTEID  CATABOLISM  [LECT.  vn. 

already  been  published  which  were  designed  to  test  the 
possibility  of  a  sugar  synthesis  from  amido  acids  or  the  oxy- 
acids  which  are  formed  from  them.  But  much  more  remains  to 
be  done  before  the  matter  becomes  clear. 

It  seems,  indeed,  that,  in  order  to  be  able  to  follow  the 
processes  of  animal  metabolism,  what  we  require  to  know  more 
than  anything  else  is  the  way  in  which  the  lower  fatty  acids  and 
their  simple  derivatives  are  oxidised.  If  we  are  right  in 
believing  that  it  is  the  oxidation  of  the  substances  obtained 
after  removal  of  the  amido  groups  from  amido  acids  that  give 
proteids  their  value  as  sources  of  energy,  then  the  reactions 
which  do  the  work  and  supply  heat  in  the  body  are  concerned 
with  similar  compounds,  whether  in  the  first  instance  it  be 
proteids,  carbohydrates,  or  fats  from  which  they  are  derived. 
The  greatest  step  forward  will  be  taken  when  we  come  to 
know  how  the  cells  dispose  of  simple  organic  compounds,  such 
as  lactic  or  acetic  acid,  what  stages  the  reactions  pass  through, 
and,  therefore,  what  side  reactions  can  occur  ;  whether  substances 
may  be  formed,  such  as  formic  or  glyceric  aldehyde,  from  which 
sugar,  or  acetic  aldehyde,  from  which,  according  to  Nencki's 
suggestion,  higher  fatty  acids  may  be  put  together.  Then  we 
shall  be  able  at  last  to  make  intelligible  the  problem  of  the 
formation  of  both  sugar  and  fat  from  proteids,  which  is  never 
likely  to  be  the  case  so  long  as  these  supremely  complex 
substances  themselves  are  all  that  we  deal  with  in  our  experi- 
ments and  discussions  on  metabolism. 


LECTURE  VIII 

THE   METABOLISM   OF  CYCLIC   FORMATIONS 

THE  greater  part  of  proteid  molecules  is  composed  of  the 
amido  and  diamido  fatty  acids,  with  which,  together  with  the 
unknown  amide  combinations,  we  were  principally  concerned 
in  the  last  lecture.  But  in  addition  to  these  there  are  a  number 
of  combinations  of  nitrogen  with  carbon  found  in  proteids,  or 
associated  with  proteids  in  the  body,  which  have  not  yet  been 
touched  upon.  And  some  of  these  are  present  in  substances 
which  have  furnished  physiology  with  not  the  least  difficult  of 
its  problems  in  metabolism. 

We  may  begin  with  the  guanidine  group,  imido  urea, 

NH2 

C  =  NH 
NH2 

which  is  found  in  arginine  combined  with  amido-valerianic 
acid  and  also  in  kreatine.  We  know  this  about  arginine,  that 
in  many  of  the  organs — the  liver,  kidney,  intestine,  thymus, 
and  lymph  glands,  but  notably  not  in  the  spleen,  pancreatic 
secretion,  or  bile — an  enzyme  is  found  which  attacks  this 
group,  removing  urea  and  leaving  the  diamido-valerianic  acid, 
ornithine.1  Besides  this  enzyme  acting  on  free  arginine,  an 

1  Kossel  and  Dakin,  H.-S.  Z.  41,  321,  and  42,  181,  1904 

165 


166       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LKCT. 

enzyme  exists  in  the  intestine  that  has  the  power  of  removing 
urea  from  the  arginine  in  one  at  any  rate  of  the  protamines, 
clupeine,  without  completely  disintegrating  it,  the  guanidine 
group  being  accessible  to  it  even  if  the  arginine  is  not  detached  ; 
so  that  after  it  has  acted,  the  clupeone,  as  the  altered  clupeine 
is  called,  gives,  when  hydrolysed  with  acids,  ornithine,  which 
was  not  the  case  before.  It  is  remarkable  that  kreatine,  in 
which  the  same  guanidine  group  is  also  attached  to  an  acid, 
is  not  acted  on  by  the  arginase :  whether  this  is  due  to  the 
methyl  group  attached  to  the  nitrogen  atom  at  which  the 
cleavage  has  to  take  place,  or  whether  it  is  because  in  acid 
media  kreatine  tends  to  pass  into  the  anhydride  form,  kreatinine, 
is  not  known.  But  we  have  in  this  fact  an  explanation  of  the 
behaviour  of  these  substances  in  the  body :  how  it  is  that  while 
boiling  with  baryta  removes  urea  from  both  kreatine  and 
arginine,  in  the  body  arginine  is  completely  destroyed, 
while  the  kreatine  that  is  taken  with  food  in  meat  passes 
through  the  body  unattacked,  and  appears  in  the  urine  as 
kreatinine. 

Kreatinine  is  formed  in  the  urine  even  when  the  food 
contains  no  kreatine.  This  endogenous  kreatinine  has  been 
shown  by  Folin  to  be  a  remarkably  constant  quantity  in  each 
individual.  In  different  people  the  amount  is  found  to  vary 
considerably,  but  for  each  person  the  amount  excreted  remains 
practically  the  same,  whether  much  proteid  is  taken  or  little, 
provided  that  the  food  contains  no  kreatine.  There  is  no  other 
nitrogenous  component  of  the  excreta  of  which  this  is  known 
to  be  true ;  so  that,  whatever  may  be  true  of  the  other  forms 
in  which  nitrogen  is  discharged  from  the  body,  the  kreatinine 
excreted  on  a  kreatine-free  diet  is  a  measure  of  the  metabolic 
decay  of  tissue.  On  a  low  proteid  diet  kreatinine  may 
account  for  nearly  20  per  cent,  of  the  whole  nitrogen 
excreted.  There  are  many  reasons  for  believing  that  it  is  in 
the  muscles  that  it  is  formed,  principally  at  any  rate.1 

1  Gregor,  H.-S.  Z.  31,  98,  1900  ;  Liebig,  Ann.  1848  ;  Monari,  M.  J.  296, 
1889;  Folin,  Am.  JL  Phys.  14,  72,  1905;  Burian,  H.-S.  Z.  43,  545, 
1905. 


viii.]  ENDOGENOUS  KREATININE  167 

Muscular  activity  is  followed  by  an  increased  output  of 
kreatinine :  more  kreatine  is  found  in  working  muscles  than  in 
those  that  have  been  at  rest:  the  amount  of  kreatinine 
excreted  is  increased  in  fevers.1  The  individual  variations  in 
the  excretion  of  this  substance  seem  to  be  determined  by  the 
mass  of  muscle.2 

How  the  kreatine  in  the  muscles  comes  into  existence  we 
do  not  as  yet  know.  If  we  attach  fundamental  importance  to 
the  guanidine  feature  of  the  molecule,  we  shall  be  especially 
interested  by  the  fact  that  Kutscher  and  Otori3  have  found 
guanidine  among  the  substances  formed  in  autolysis  of  the 
pancreas,  in  traces  also  among  the  products  of  hydrolysis  of 
pseudo-mucin,  and  also  that  it  is  formed  when  certain  proteids 
are  oxidised  by  permanganate ;  in  this  case  in  greater  quantity 
than  could  be  accounted  for  by  supposing  it  to  be  derived  from 
the  arginine  in  those  proteids.  And  that  may  lead  to  the 
suspicion  that  guanidine  groups  other  than  that  of  arginine  are 
present  in  proteids  generally,  the  muscle  proteids  more  than 
the  rest,  and  that  the  kreatine  formed  in  muscles  is  a  fragment 
of  the  proteid  molecule,  which  in  the  process  of  separation  has 
itself  undergone  but  little  change.  Or,  on  the  other  hand,  it 
is  possible  that  the  guanidine  is  an  acquired  characteristic  of 
the  compound  ;  that  kreatinine  is  the  more  fundamental  form 
with  the  ring  structure,  shown  in  its  formula, 

CH3.N CH2 

NH .  C         CO 

v 

NH 

and  that  this  ring  structure  gives  rise  to  the  guanidine  con- 
figuration, incidentally,  as  it  were,  when  its  ring  is  opened. 
This  is  a  point  to  which  we  may  return  later. 

1  Moritz,   D.   A.  /.  k.  M.  46;    Hofmann,   V.  A.  48,    1869;   Salkowski, 
V.  A.  88,  391.     According  to  van  Hoogenhuyze  and  Verploegh,  ff.-S.  Z. 
46,  415,  1905,  it  is  only  in  starvation  that  muscular  exertions  increase  the 
output  of  kreatinine  appreciably. 

2  Folin,  Am.  Jl.  Phys.,  xiii.,  85,  1905. 

3  Kutscher  and  Otori,  H.-S.  Z.  42,  453,  and  43,  98,  1904. 


168        THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

In  any  case,  it  is  important  to  remember  that  the  kreatinine 
in  the  urine  is  probably  a  measure  of  physiological  decay  of 
muscular  tissue.  It  is  a  substance  particularly  rich  in  nitrogen, 
which  forms  37  per  cent,  of  its  weight,  and  so  may  account  for 
a  large  proportion  of  the  nitrogen  waste  of  the  muscles.  Urea, 
at  any  rate,  is  formed  in  them  to  a  very  small  extent  in  com- 
parison with  the  body  generally :  to  what  extent  uric  acid  is 
also  produced  in  the  metabolism  of  muscle,  we  may  leave  for 
consideration  later. 

One  further  point  with  regard  to  kreatine  may  be  noted 
here :  the  presence  of  a  methyl  group  attached  to  nitrogen, 
constituting  it  a  methylamine  derivative,  is  remarkable.  Choline 
and  its  derivatives,  and  adrenaline,  are  the  only  other  well- 
known  instances  of  this  combination  in  the  body.  But  the 
methyl  mercaptan  found  by  Nencki1  in  normal  urine  in 
traces,  and  in  larger  amounts  after  taking  asparagus,  and 
the  excretion  of  tellurium  2  as  methyl  tellurium  after  admini- 
stration of  tellurous  acid,  and  the  methylation  of  xanthine 
to  heteroxanthine 3  (y-methyl  xanthine)  in  the  dog,  are 
curiosities  in  this  respect  related  to  the  methylamine 
derivatives. 

The  other  nitrogenous  combinations  found  in,  or  associated 
with  proteids,  are  all  of  them  heterocyclic  ring  formations  ;  rings, 
that  is,  in  which  some  of  the  atoms  are  not  carbon  atoms.  The 
simplest  of  these  rings  is  that  found  in  pyrrhol — 

HC CH 

Hi 


Pyrrhol  is  formed  when  proteids  are  heated  with  baryta  to 
1 50°  C.  But  the  first  simple  derivative  of  pyrrhol  to  be  iso- 
lated and  identified  among  substances  formed  from  proteids 

1  Nencki,  S.  A.  28,  206,  1891. 

2  Hofmeister,  S.  A.  33,  198,  1894. 

3  Neuberg,  SalowskPs  Festschrift,  1904. 


]        PROLINE,  ARGININE,  AND  GLUTAMIC  ACID        169 

under    physiological    conditions    was    pyrrholidine    carboxylic 
acid — 

H9C Ct*2 

H2C         CH .  COOH 


— proline,  as  it  is  called  for  short,  which  Emil  Fischer l  first  found 
among  the  products  of  hydrolysis  of  casein  and  other  proteids, 
in  amounts  varying  from  about  5  per  cent,  in  gelatine  to  3  per 
cent  in  casein,  and  1.5  per  cent,  in  egg-albumin  and  haemo- 
globin. The  question  was  at  once  raised  by  Fischer  himself, 
whether  this  new  compound  was  not  formed  by  a  secondary 
reaction  in  the  course  of  the  esterification  process  by  which  it 
was  obtained  ; — either  arginine  or  diamido-valerianic  acid  could 
conceivably  have  given  rise  to  it,  but  neither  of  these  substances, 
treated  by  themselves  in  the  same  way  as  that  in  which  the 
mixed  products  of  proteid  hydrolysis  had  been  treated,  yielded 
anything  that  behaved  like  proline.2  He  has  obtained  it  from 
casein  by  boiling  with  alkalies  instead  of  acids  ;3  but  though  it  was 
not  liberated  by  tryptic  digestion,  a  polypeptide  which  resisted 
tryptic  action  for  seven  months,  a  substance  precipitated  by  phos- 
photungstic  acid  but  not  showing  the  biuret  reaction,  was  found, 
which,  when  boiled  with  acids,  gave  up  all  the  proline  as  well 
as  the  phenyl-alanine  that  could  be  obtained  from  casein  by 
direct  hydrolysis.4  Peptic  digestion,  on  the  other  hand,  especially 
if  followed  by  tryptic,  hydrolyses  the  greater  part  of  this  com- 
pound in  which  the  proline  is  contained,  and  under  these 
circumstances  the  proline  can  be  isolated  without  exposing  the 
mixture  in  which  it  is  found  to  the  action  of  hot  acids  at  any 
stage  of  the  process.5  Fischer  concludes,  therefore,  that  the 
•proline  is  as  much  a  primary  product  of  proteid  hydrolysis  as 
leucine  itself.  It  is  even  possible  that  it  may  prove  to  be  the 
parent  substance  from  which  other  constituents  of  the  proteid 
molecule  are  descended,  for  another  derivative,  an  oxyproline,6 

1  E.  Fischer,  H.-S.  Z.  33,  152,  1901.  2  Id.,  p.  170. 

3  H.-SZ.  35,  227,  1902.  4  H.-S.  Z.  39,  81,  1903- 

5  ff.-S.  Z.  40,  216,  1903.  6  H.-S.  Z.  39,  155. 


170       THE  METABOLISM  OF  CYCLIC  FORMATIONS      [LKCT. 

has  been  found  by  Fischer  in  casein  and  gelatine.  The  exact 
position  of  the  hydroxyl  group  has  not  been  determined,  but  if 
it  should  prove  to  be  in  the  S  position, 


CH0 CH2 

f  I 

HO  .  CH          CH  .  COOH 


then  it  may  be  evidence  for  the  intramolecular  oxidation  of 
the  pyrrhol  ring  to  glutamic  acid.  From  glutamic  acid  a 
pyrrholidone  carboxylic  acid l  has  been  obtained, 


CH2 CH. 


CH .  COOH 

\/ 

NH 

and  Fischer  came  across  this  latter  substance  among  those 
formed  in  the  hydrolysis  of  horn,  but  preferred  to  regard  it  as 
a  secondary  product  derived  from  glutamic  acid.  The  possibility 
of  a  close  relationship  between  proline  and  glutamic  acid,  so 
long  as  it  remains  a  possibility,  adds  to  the  physiological  interest 
of  this  pyrrhol  derivative. 

Another  substance,  unquestionably  of  first-rate  importance, 
in  which  the  pyrrhol  ring  occurs  is  haematine.  Pyrrhol  is 
formed  when  haematine  is  subjected  to  dry  distillation,2  and 
also,  as  Nencki  showed,  when  it  is  reduced  with  tin  and  hydro- 
chloric acid  and  distilled  with  alkali,  or  when  it  is  fused  with 
potash.3  No  less  important  in  vegetable  physiology  than 
haematine  in  the  physiology  of  animals  is  chlorophyll,  and  the 
phylloporphyrine  obtained  from  chlorophyll  by  Schunck  and 
Marchlewski,  which  they  proved  to  differ  in  its  elementary 
composition  from  haematoporphyrine  only  in  containing  two 
atoms  of  oxygen  less,  and  spectroscopically  to  be  almost 
indistinguishable  from  it,  like  haematoporphyrine  also  gives 

1  M.f.  Ch.  3,  228.     2  Hoppe-Seyler,  Med.  chem.  Untersuchungen,  p.  523. 
3  Nencki  and  Sieber,  S.  A.  418,  1884. 


VIII.] 

pyrrhol  on  distillation  with  zinc  dust.1  Nencki  and  Zaleski2 
then  obtained  a  further  link  connecting  these  two  pigments, 
by  preparing  from  haemine  or  haematoporphyrine  on  reduction 
with  phosphonium  iodide  two  substances :  mesoporphyrine,  with 
the  formula  C16H18N2O2,  coming,  therefore,  between  haemato- 
porphyrine, C16H18N2O3,  and  phylloporphyrine,  C16H18N2O  ;8  and 
besides  mesoporphyrine,  haemopyrrhol,  which  was  also  obtained 
by  Marchlewski  and  Nencki  from  phylloporphyrine.4  This 
haemopyrrhol,  then,  with  the  formula  C8H13N,  is  a  common 
derivative  of  both  chlorophyll  and  haematine,  and  from  which- 
ever source  it  is  obtained,  it  is  oxidised  on  exposure  to  the  air 
to  urobiline.  For  haemopyrrhol  Nencki  and  Zaleski  suggested 
two  possible  formulae,  of  which  one,  that  of  a  methyl  propyl 
pyrrhol, 

CH3.C C.C3Hr 


has  been  shown  to  be  probably  correct.  Kuster5  oxidised 
haematine  or  haematoporphyrine  with  bichromate  in  glacial 
acetic  acid,  and  obtained  a  substance,  a  haematinic  acid  with 
the  formula  C8H9NO4,  which  on  dry  distillation  also  gave 
pyrrhol,  and  when  treated  with  alkali  gave  up  ammonia,  and 
was  converted  into  a  second  haematinic  acid,  which  differed 


from  the  other  by  having  exchanged  an  NH  group  for  an  atom 
of  oxygen.  The  relation  of  these  two  acids  was  clearly  to  be 
expressed  by  the  formulae 

RC==CR'  RC=  =  CR' 

CO         CO         and  CO         CO 

V 

1  Schunck  and  Marchlewski,  Ann.  278,  284,  288,  and  290,  1896. 

2  Nencki  and  Zaleski,  B.  34,  997,  1901. 

3  These  formulae  are  not  final ;  cf.  Zaleski,  H.-S.  37,  73,  1902. 

4  Marchlewski  and  Nencki,  B.  34,  1687,  1901. 

5  Kuster,  H.-S.  Z.  28,  i,  1899  ;  and,  44,  391,  1905. 


172       THE  METABOLISM  OF  CYCLIC  FORMATIONS      [LECT. 

the  imide  and  anhydride  respectively  of  the  same  tribasic  acid. 
The  value  of  R  and  R'  in  these  formulae  was  determined  by 
the  fact  that  the  anhydride  on  further  oxidation  gave  succinic 
acid,  COOH  .  CH2 .  CH2 .  COOH,  and  also  could  be  converted  by 
the  loss  of  CO2  into  a  substance  identical  with  methyl  ethyl 

maleic  anhydride — 

CH3.C  =  C.C2H5 

CO         CO 

\/ 
O 

The  value  of  R,  therefore,  is  CH3,  and  that  of  R'  is 
CH2.  CH2.  COOH  ;  and  the  constitution  of  haemopyrrhol  is 
almost  proved  to  be 

CH3.C C.C3Hr 


H.i 


NH 


C.H 


—  methyl  propyl  pyrrhol. 

Its  synthesis  by  reduction  of  methyl  propyl  maleic  imide  has 
indeed  been  already  attempted,  though  the  results  are  not  con- 
clusive as  yet.1  It  is,  therefore,  beginning  to  be  possible  to  look 
forward  to  the  synthesis  of  the  blood  pigments,  and  to  the 
opening  up  of  entirely  new  prospects  over  the  field  of  biological 
chemistry. 

The  pyrrhol  formation  occurs  in  another  important  combina- 
tion.    Indol,  with  the  formula, 
CH 

S 
HC 


may  be  regarded  as  compounded  of  benzene  and  pyrrhol 
grafted  on  to  each  other  so  as  to  share  two  carbon  atoms  in 
common.  All  the  known  derivatives  of  indol  occurring  in  the 
body  can  be  traced  to  one  and  the  same  origin,  one  of  the 
1  Marchlewski  and  Buraczewski,  /T.-5.  Z.  43,  410,  1905. 


viii.]  TRYPTOPHANE:  ITS  CONSTITUTION  173 

compound  amido  acids  that  enters  into  the  composition  of 
proteids,  with  but  few  exceptions  such  as  gelatine,  and  is  set 
free  by  the  hydrolytic  action  both  of  acids  and  of  trypsine, 
from  the  latter  of  which  properties  it  received  its  name  of 
tryptophane  long  before  it  was  identified.  The  constitution  of 
this  substance  has  not  been  precisely  determined.  Hopkins 
and  Cole,1  who  first  isolated  and  described  it,  found  that  the 
substances  produced  by  bacteria  in  the  intestine,  known  as 
scatol  acetic  and  carboxylic  acids,  and  scatol  itself,  were  formed 
by  bacteria  from  tryptophane,  which  they-  therefore  regarded  as 
scatol  amido-acetic  acid.  But  it  has  since  been  shown 2  that  the 
substance  which  had  been  regarded  as  scatol  carboxylic  acid  is 
really  indol  acetic  acid.  For  it  is  identical  with  the  product  of 
the  action  of  zinc  chloride  upon  the  phenyl  hydrazone  of  aldehydo- 
propionic  acid, 

6    5\N.N:CH.CH2.CH2.COOH, 
H/ 

condensation  taking  place  thus— 

C6H4 C.CH2.COOH 

NH CH 

The  acid,  therefore,  known  as  scatol  acetic  must  really  be  indol 
propionic,  and  tryptophane  be  indol  amido-propionic  acid.  But 
Ellinger  made  another  interesting  contribution  to  the  chemistry 
and  physiology  of  tryptophane  by  discovering  that  when 
administered  to  dogs  it  was  converted  into  kynurenic  acid,  which 
he  found  in  the  urine.3  Now  this  substance  was  known  to  have 
the  constitution  represented  in  the  formula 

OH 

COOH 


N 

1  Hopkins  and  Cole,//,  of  Phys.  27,  418,  and  29,451,  1903. 

2  Ellinger,  B.  37,  1801,  1904. 

3  Ellinger,  H.-S.  Z.  43,  325,  1904. 


174       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT 

The  conversion  of  an  indol  amido-propionic  acid  into  a  body 
of  this  constitution  is  difficult  to  imagine.  Ellinger  attempted 
to  solve  the  difficulty  by  ascribing  to  tryptophane  the  constitu- 
tion a-indol  ^-amido-propionic  acid,  and  represented  the  change 
graphically  thus : 

N_ 

V  COOH 


OH 

\/ 


But  it  is  a  question  whether  this  is  the  only  or  the  most 
probable  way  in  which  the  change  could  occur,  and  it  is, 
therefore,  not  necessary  to  regard  tryptophane  as  a  /3-amido 
acid.  The  amido  acids  that  occur  in  Nature  are  almost  without 
exception  the  a-acids,  and  it  is  questionable  whether  there  is 
any  justification  for  the  supposition  that  in  tryptophane  we 
have  an  exception  to  this  rule.  The  only  instance  of  a  /3-amido 
acid  in  the  chemistry  of  living  organisms  is  the  iso-cystine 
described  by  Neuberg  and  Mayer.1  This  is  a  cystine  which 
they  found  in  the  urinary  calculi,  differing  in  several  respects 
from  that  which  is  obtained  from  proteids  and  from  that  found 
in  the  urine  of  cystinuric  patients,  so  far  as  we  know  at  present. 
This  iso-cystine  they  think  is  derived  from  /3-amido  ce-thiolactic 
acid  instead  of  the  a-amido  /5-thiolactic  acid  which  is  ordinary 
cysteine  :  from 

CH2.NH2  HS.CH2 

HS .  CH  and  not  from  CH  .  NH2 

COOH  COOH 

Morner,2  also,  from  a  study  of  the  cystine  obtained  from 
proteids,  came  to  the  conclusion  that  these  two  kinds  of  cysteine 
entered  into  its  composition.  For,  when  he  heated  it  under 

1  Neuberg  and  Mayer,  H.-S.  Z.  44,  472,  1905  ;  cf.  ff.-S.  Z.  43,  338,  1904. 

2  Morner,  H.-S.  Z.,  p.  363,  1904. 


viii.]  ISO-CYSTINE  175 

pressure  with  hydrochloric  acid,  besides  ammonia  and  sulphu- 
retted hydrogen,  he  found  that  both  a-amido-propionic  acid  and 
a-thiolactic  acid  were  formed.  Now  both  Neuberg  and 
Friedmann l  have  made  it  clear  that  in  cystine  the  sulphur  and 
the  nitrogen  are  united  to  different  carbon  atoms,  and  not  the 
same  one  as  Baumann  supposed.  In  the  molecule,  therefore, 
from  which  a-thiolactic  acid  was  formed,  the  amido  group,  he 
argues,  must  have  been  in  the  (3  position.  The  occurrence  side 
by  side  of  these  two  cysteines  would  be  most  easily  intelligible 
on  the  supposition,  proposed  by  Neuberg,2  that  they  were  both 
formed  from  the  same  substance, 

COOH 


:i 


HS.CH 
NH2CH 

COOH, 

by  the  removal  of  CO2  from  the  two  different  ends  of  the 
molecule :  just  as  from  the  corresponding  oxy-aspartic  acid, 
which  has  actually  been  obtained  by  Skraup3  from  casein,  it 
may  be  expected  that  in  addition  to  serine,  /3-oxy  a-amido- 
propionic  acid,  the  iso -serine  with  the  hydroxyl  and  amido 
groups  transposed,  which  has  been  synthesised  by  Fischer  and 
Leuchs,4  should  be  formed.  But  the  iso-cystine  found  by 
Neuberg  and  Mayer  is  at  any  rate  unusual,  even  in  cystine 
stones,  and  the  actual  sample  they  prepared  was  not  free  from 
tyrosine,  or  at  any  rate  gave  Millon's  reaction,  as  Fischer5 
showed ;  and  Fischer  suggests  that  this  impurity  may  possibly 
account  for  some  of  the  peculiarities  of  the  cystine,  which  were 
the  only  ground  for  supposing  it  to  contain  the  amido  group  in 
the  /3  position. 

We  do  not,  therefore,  positively  know  of  any  /3-amido  acid 

1  Neuberg,  P.  35,  3161,  1901  ;  Friedmann,  H.  B.  3,  I5  and  184,  1902. 

2  Neuberg,  H.-S.  Z.  43,  340. 

3  Skraup,  H.-S.  Z.  42,  274,  1904. 

4  Fischer  and  Leuchs,  B.  35,  3790,  1902. 

5  Fischer  and  Suzuki,  H.-S.  Z.  45,  410,  1905. 


176       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

in  Nature,  and  are  perhaps  justified  in  hesitating  to  accept  a 
constitutional  formula  for  tryptophane  which  has  this  anomaly.1 
But,  however  we  may  have  to  account  for  the  formation  of 
kynurenic  acid  from  tryptophane,  the  possibility  of  the  deriva- 
tion of  a  quinoline  compound  from  this  group,  which  is  present 
in  all  typical  proteids,  is  one  of  great  interest,  and,  as  Ellinger 
suggests,  it  is  possible  that  the  alkaloids  with  a  quinoline  or 
pyridine  nucleus  that  occur  in  plants  may  be  derived  from 
tryptophane  by  similar  reactions  in  vegetable  metabolism. 

The  part  played  by  tryptophane  in  animal  metabolism  is 
obscure.  It  is  well  known  that  in  the  intestine  it  is  set  free  by 
the  pancreatic  juice,  and  that  bacteria  in  the  intestine  decom- 
pose it  in  so  far  as  it  is  not  absorbed  unchanged.  In  this 
decomposition,  however,  it  is  the  side  chain  alone  that  is 
attacked ;  denitrification  gives  rise  to  indol  propionic  acid, 
oxidation  of  this  to  indol  acetic  acid,  and  this  losing  CO2 
becomes  scatol,  or  by  oxidation  nearer  the  chain,  and  further 
loss  of  CO,  indol  itself.2  Of  these  products  of  bacterial  change, 
indol  acetic  acid  may  be  found  in  the  urine,3  and  indol  and 
scatol  oxidised  to  the  corresponding  phenol  and  combined  with 
glycuronic  or  sulphuric  acid  are  constantly  found  in  small 
amount  in  human  urine.  But  the  principal  part  of  these 
changes  is  the  work  of  bacteria,  and  not  the  metabolism  of  the 
body  itself.  Tryptophane  is  contained  in  the  proteids  which 
break  down  in  metabolism,  and  is  set  free,  not  only  by  trypsine, 
but  by  the  cellular  enzymes.  Does  this  endogenous  tryptophane 
and  the  tryptophane  that  is  absorbed  unchanged  from  the 
intestine  undergo  the  same  changes  in  the  cells  as  the  bacteria 
bring  about  in  the  intestine?  If  so,  does  the  indol  ring  under 
these  conditions  escape  into  the  urine  unbroken,  and  contribute 
to  the  urinary  indican?  This  is  a  question  which  has  been 

1  More  recently  Ellinger  has  synthesised  the  acid  formerly  known  as 
scatol  acetic  acid,  and  from  the  synthesis  it  is  clear,  not  only  that  this  acid 
is  indol  propionic  acid,  but  that  the  indol  is  attached  to  the  ^-carbon  atom, 
and  not  as  in  Ellinger's  suggested  formula  for  tryptophane  to  the  a  atom. — B. 
38,  2884,  1905. 

2  Salkowski,  H.-S.  Z.  9,  27,  1885.  » Ibid. 


viir.]  ORIGIN  OF  URINARY  INDICAN  177 

warmly  disputed.  It  was  taught  till  recently  that  all  the  indican 
in  the  urine  was  the  result  of  bacterial  decomposition.  Jaffe l 
showed  how  obstruction  in  the  small  intestine  in  dogs  caused  a 
great  increase  in  the  indican  excreted  ;  obstruction  of  the  large 
intestine,  a  much  smaller  increase,  if  any — clinical  experience  in 
man  agrees  with  this.  Ellinger  and  Prutz2  have  shown  that 
the  obstruction  caused  by  anti-peristalsis,  induced  by  dividing 
the  small  intestine  in  two  places  a  few  inches  apart  and  joining 
the  intervening  length  of  intestine  on  again  the  wrong  way 
round,  caused  the, indican  excretion  to  be  increased  enormously 
— twenty  and  thirtyfold.  No  such  pronounced  alteration  as  this 
is  known  to  be  set  up  in  any  way  in  which  the  operation  of 
intestinal  decomposition  can  be  excluded.  Recently,  however, 
Blumenthal 3  has  maintained  that  some  of  the  urinary  indican 
is  of  a  different  origin.  Rabbits,  under  ordinary  conditions, 
excrete,  he  says,  no  indican,  but  in  starvation,  when  they  are 
living  on  their  tissues,  indican  is  found  in  their  urine.  Diabetic 
puncture  in  these  animals  also  brings  on  indicanuria,  and  in 
man  he  traces  certain  cases  of  marked  indicanuria  to  nervous 
disturbances.  The  increased  proteid  destruction  set  up  by 
phlorrhizine  in  rabbits  also  is  accompanied  by  indicanuria.4 
But  the  indican  excreted  by  starving  rabbits  is  differently 
explained  by  Ellinger.5  These  animal's,  when  food  is  withheld, 
begin  almost  on  the  first  day  to  consume  their  own  dejecta, 
and  if  by  effectual  muzzling  they  are  prevented  from  doing 
this,  the  indicanuria  is  not  observed.  The  experiments  with 
phlorrhizine,  repeated  by  Scholtz,6  gave  negative  results.  So 
that  the  view  that  some  of  the  indican  in  the  urine  is  formed  in 
tissue  metabolism  is  not  well  supported  by  evidence.  As  a 
matter  of  fact,  subcutaneous  injection  of  tryptophane  in  rabbits 
and  dogs  does  not  increase  the  indican  in  the  urine,7  while 

1  Jaffe,  V.  A.  70,  p.  77,  1870. 

2  Ellinger  and  Prutz,  U.-S.  Z.  38,  400,  1903. 

3  Blumenthal,  M.  /.,  p.  817,  1902.        4  Lewin,  H.  B.  i,  493,  1902. 

5  Ellinger,  ff.-S.  Z.  39,  52,  1903.         6  Scholtz,  H.-S.  Z.  38,  513,  1903. 
7  Ellinger  and  Gentzen  ,//.  B.,  4,  174,  1903;  Rosenfeld,  H.  B.,  v.,  83, 
1903. 

M 


178       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

subcutaneous  injections  of  indol  does.1  So  that  it  is  not  likely 
that  tryptophane  is  dealt  with  by  the  cells  in  the  body  in  the 
same  way  as  it  is  by  the  bacteria  in  the  intestine,  or,  in  other 
words,  it  is  improbable  that  the  breakdown  of  tryptophane  in 
metabolism  leads  to  the  liberation  of  indol.  In  some  animals, 
at  any  rate  rabbits  and  dogs,  it  is  partially  changed  into 
kynurenic  acid,2  but  the  bulk  of  it  apparently  is  oxidised,  and 
the  indol  ring  itself  is  not  spared. 

Closely  related  to  pyrrhol  is  imidazol,  which  is  also  a  ring  of 
five  atoms,  but  of  these  five,  two  are  nitrogen  : 

HC NH 

\CH 
HC N 

The  corresponding  6-atom  ring,  containing  one  additional  carbon 
atom,  is  pyrimidine, 

N  =  CH 


HC 


CH 


in  which  it  is  customary  to  number  the  several  atoms,  beginning 
from  the  nitrogen  at  the  top  of  the  above  molecular  diagram, 
as  No.  I,  working  down  the  left-hand  column  and  then  up  the 
right-hand  column  to  the  carbon  No.  6  at  the  top  on  the  right. 

These  two  rings  are  most  familiar  to  us  in  the  form  of  the 
compound  ring  that  results  from  their  fusion,  and  is  purine. 
Just  as  indol  may  be  regarded  as  benzene  grafted  on  to  pyrrhol, 
purine  is  pyrimidine  grafted  upon  imidazol. 

Since  both  imidazol  and  pyrimidine  rings  occur  in  substances 
of  physiological  importance,  they  may  be  dealt  with  individually 
before  we  pass  to  the  more  familiar  purine. 

Imidazol  has  recently  been  proved  to  enter  into  the  forma- 


1  Grosser,  H.-S.  Z.  44,  320,  1905. 

2  Ellinger,  H.-S.  Z.  43,  325,  I9°4- 


viii.]  IMIDAZOL  DERIVATIVES  179 

tion  of  histidine.  This  base,  first  described  by  Kossel,1  who 
obtained  it  by  hydrolysing  the  protamine  sturine,  was  shown 
by  Hedin2  to  be  present  in  a  large  number  of  proteids.  Its 
empirical  formula,  C6H9N3O2,  showed  that  it  could  not  be  built 
on  a  saturated  open  chain  of  carbon  atoms,  as  the  amount  of 
hydrogen  was  too  small.  Certain  facts  with  regard  to  its  con- 
stitution came  to  light  from  time  to  time,3  but  a  correct 
inference  as  to  the  arrangement  of  the  atoms  in  the  molecule 
was  first  made  by  Pauly,4  who  pronounced  for  a  compound  of 
imidazol  and  alanine.  This  was  proved  to  be  correct  by  Knoop 
and  Windaus,  who  synthesised  imidazol  propionic  acid,  and 
showed  that  it  was  the  same  substance  as  that  formed  from 
histidine  by  substituting  hydroxyl  for  the  amido  group  and 
then  reducing  the  product.5  Histidine,  therefore,  like  tyrosine, 
phenyl-alanine,  cysteine,  tryptophane,  and  serine,  is  a  compound 
of  amido-propionic  acid. 

But  histidine  is  not  the  only  substance  familiar  in  physiology 
in  which  the  imidazol  ring  occurs.  Kreatinine  is  also  built  up 
upon  this  plan,  as  is  seen  from  its  formula — 

CH2 N/ 

\C  =   NH 
CO NH 

and  it  is  possible  that  this  may  furnish  a  clue  as  to  the  mode  of 
its  formation  in  muscle.  And  the  interest  attaching  to  such 
imidazol  derivatives  has  been  greatly  increased  by  the  remark- 
able fact  observed  by  Knoop  and  Windaus,6  that  out  of  glucose, 
in  the  presence  of  ammonia  and  zinc  oxide,  methyl  imidazol  is 
formed  in  large  quantities  under  the  influence  of  light.  This 
they  suppose  to  take  place  through  the  condensation  of  pyruvic 

1  Kossel,  H.-S.  Z.  22,  176,  1896. 

2  Hedin,  H.-S.  Z.  22,  191,  1896. 

3  Herzog,  H.-S.  Z.  37,  248  ;  Frankel,  M.  /.,  p.  152,  1903. 

4  Pauly,  H.-S.  Z.  42,  508. 

6  Knoop  and  Windaus,  H.  B.  7,  144,  1905. 

6  Knoop  and  Windaus,  B.  38,  5  ;  H.  B.>  vi.,  392,  1905. 


180        THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 


aldehyde  with  two  molecules  of  ammonia  and  one  of  formal- 
dehyde— 
CH, 

CH3 
:=O     +   NH3  ,H 

+     C   =   O >      C NH 


NHf 


CH 


HC N 

Pyruvic  aldehyde  has,  as  we  have  seen,  been  regarded  as  one 
of  the  probable  intermediate  products  in  the  formation  of  lactic 
acid  from  sugar.  They  point  out  that  the  methyl  imidazol 
formation  occurs  in  purine,  as  is  best  seen  from  the  formula  for 
xanthine — 

NH— CO 

CO  C— NHv 

NH— C  —  N  ^ 

which  is  an  ureide  of  imidazol  carboxylic  acid. 
The  simple  derivatives  of  pyrimidine, 

N1=6CH 

H .  C2     5CH 

N3    4CH 

that  have  been  obtained  from  the  animal  body  have  all  of  them 
been  separated  from  the  products  of  hydrolysis  of  nucleic  acids. 
If  nucleic  acids  are  heated  under  pressure  with  acids,  or  boiled 
for  twelve  hours  or  more  with  30  to  40  per  cent,  sulphuric  acid, 
one  or  more  of  the  following  substances  are  found  to  have  been 
split  off — uracil,  thymine,  or  cytosine.  The  nucleic  acid  of 
herrings'  sperma  gives  all  three  of  them.  Thymine  has  not 
been  obtained  from  nucleic  acids  of  vegetable  origin ;  nucleic 
acids  of  animal  origin  give  one  or  other,  generally  two,  of  these 
bodies.  Uracil  has  been  synthesised  by  Fischer  and  Roeder  x 

1  Fischer  and  Roeder,  B.  34,  3751,  1901. 


viii.]  PYRIMIDINE  DERIVATIVES  181 

from  acrylic  acid  and  urea  by  removing  two  hydrogen  atoms 
from  the  dihydro  uracil  first  formed  when  these  two  substances 
condense,  and  thymine  in  the  same  way  was  obtained  by  them 
from  urea  and  methyl  acrylic  acid.  Their  constitution  is 
therefore  represented  thus : 

HN— CO 

OC    CH 

I       II 
HN— CH 

Uracil  =  2.6-Oxypyrinidine.  Thymine  =  5-M ethyl  uracil. 

Cytosine  has  also  been  synthesised  by  Wheeler  and  Johnson,1 
and  its  constitution  therefore  finally  determined.  It  is  2-oxy 
6-amido  pyrimidine, 

N=C.NH2 

O=C        C .  H 

I          II 
HN C  .  H 

The  question  whether  these  pyrimidine  derivatives  are 
actually  as  such  contained  in  the  nucleic  acids  from  which  they 
have  been  obtained,  or  whether  they  are  derived  from  the 
purine  bases  of  the  nucleic  acids,  has  been  discussed  but  not 
conclusively  settled.  The  opinion  of  those  who  have  worked 
most  on  these  bodies  seems  to  be,  generally,  that  the  pyrimidine 
nucleus  is  present  in  the  nucleic  acids  in  the  simple  form  as 
well  as  in  the  form  of  purine.2  And  in  the  case  of  thymine, 
at  any  rate,  the  methyl  group  does  not  correspond  to  anything 
in  any  of  the  purine  bases.  But  much  more  energetic  treatment 
is  necessary  to  split  them  off  than  is  usually  necessary  in 
hydrolysis,  and  though  the  purine  bases,  uncontaminated  with 
other  substances  when  treated  in  this  way,  are  not  decomposed 
so  as  to  yield  pyrimidine  derivatives,  it  is  possible  that  they 

1  Wheeler  and  Johnson,  Am.  Ch.  Jl.  29,  p.  492,  1903. 

2  Osborne  and  Harris,  H.-S.  Z.  36,  109 ;  Kossel  and  Stendel,  H.-S.  Z. 
38,  49- 


182       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

might  be  so  decomposed  in  the  presence  of  the  carbohydrate 
and  other  constituents  of  the  nucleic  acids.1 

Purine  may,  as  we  have  seen,  be  considered  as  a  compound 
ring  in  which  imidazol  is  grafted  on  pyrimidine.  And  it  may 
be  as  well  to  bear  this  conception  of  the  structure  of  purine  in 
mind.  It  is  true  that  nothing  that  is  at  present  known  points 
to  this  relationship  having  any  physiological  significance.  But, 
then,  at  present  we  do  not  know  how  the  purine  bases  come  into 
being  in  the  body,  as  they  certainly  may.  Young  animals,  during 
suckling  at  any  rate,  take  up  from  the  milk  something  which  is 
not  purine,  from  which  they  make  these  bases,  and  the  increase 
in  the  amount  of  bases  in  their  bodies  is  about  proportional  to 
the  increase  in  weight.2  A  synthesis  of  purine  must  also  take 
place  in  the  developing  chick,  since  eggs  contain  no  purine.  It 
has  been  suggested  by  Knoop  and  Windaus,  though  as  yet 
there  is  no  direct  evidence  for  the  suggestion,  that  the  methyl 
imidazol  which  can  be  formed  from  sugar  and  ammonia  in 
sunlight  could,  after  oxidation  of  the  methyl  group,  condense 
with  urea  and  so  form  xanthine.  The  possibility  of  a  simple 
synthesis  of  a  ring  so  closely  related  to  purine  from  just  the  sort 
of  material  that  would  be  available,  is  at  any  rate  interesting. 

The  question  of  the  origin  of  purine  in  the  body  introduces  the 
whole  of  what  is  still  one  of  the  most  difficult  and  vexed 
questions  in  metabolism — -the  origin  and  meaning  of  uric  acid  in 
the  body.  It  was  difficult,  in  the  days  before  the  chemical 
constitution  and  relationships  of  uric  acid  had  been  cleared  up, 
and  before  the  distribution  and  physiological  importance  of  the 
xanthine  bases  in  the  nuclei  of  all  cells  had  been  appreciated. 
But  even  now  that  these  advances  have  been  made,  the  subject 
remains  a  difficult  one,  and  at  the  same  time  has  gained  in 
significance.  It  divides  itself  now  into  a  number  of  chapters. 
Uric  acid  is  formed  in  the  body  from  related  substances  which 
are  present  free  or  combined  in  the  food ;  this  is  known  as  the 
exogenous  uric  acid.  It  is  supposed  to  be  formed  from  similar 
substances,  constituents  of  cells  in  the  body,  set  free  when  the 

1  Cf.  Burian,  Ergeb.^  vol.  iii.,  p.  99. 

2  Burian  and  Schur,  ff.-S.  Z.  23,  55,  1897. 


viii.]  OXIDATION  OF  PUR1NE  BASES  183 

cells  perish,  or  when,  as  a  result  possibly  of  their  activity,  they 
give  up  certain  components  of  their  nuclei.  And  this  endo- 
genous uric  acid,  like  the  exogenous  uric  acid,  is  formed  by  the 
oxidation  of  less  highly  oxidised  derivatives  of  purine.  But 
besides  this,  in  birds  and  reptiles  certainly,  the  purine  ring  is 
put  together  from  substances  constitutionally  not  related  to  it, 
all  forms  of  nitrogenous  combinations  contributing  nitrogen  for 
the  elaboration  of  this  highly  complex  excretory  substance,  and 
there  are  grounds  for  thinking  that,  to  some  extent  at  any  rate, 
this  synthesis  takes  place  in  mammalian  animals  and  man.  This 
question,  then,  has  to  be  considered,  whether  uric  acid  is  ever 
made  from  material  that  has  not  already  the  purine  stamp  upon 
it ;  and  then  there  still  remains  the  question  the  converse  of  this, 
whether  whatever  has  the  shape  of  purine  is  necessarily 
indestructible,  and  though  it  may  be  oxidised  to  uric  acid,  must 
then  be  excreted  as  uric  acid,  being  incapable  of  undergoing 
changes  by  which  it  would  cease  to  be  purine  at  all ;  whether 
variations  in  the  amount  of  uric  acid  excreted  in  man,  for 
instance,  may  not  depend  on  variations  in  the  activity  of  normal 
processes  in  which  the  purine  ring  is  split,  and  uric  acid  or  its 
immediate  predecessors  destroyed. 

Under  these  four  headings  may  be  grouped  most  of  what 
has  recently  been  done  to  advance  our  knowledge  of  this  side  of 
the  nitrogenous  metabolism  of  the  body. 

To  begin  with  the  exogenous  uric  acid,  it  is  now  universally 
acknowledged  that  uric  acid  is  formed  from  the  purine  bases, 
free  and  combined,  present  in  the  food.  But  this  has  not  been 
long  established.  It  was  some  years  after  Miescher  and  Kossel 
had  indicated  the  significance  of  the  wide  distribution  of 
xanthine  bases  and  their  presence  in  all  nuclei,  that  v.  Mach  and 
Minkowski1  showed  that  in  birds,  after  the  removal  of  the  liver, 
hypoxanthine  given  by  the  mouth,  unlike  most  nitrogenous 
compounds,  was  still  converted  into  uric  acid.  The  power  of 
synthesising  uric  acid  was  to  a  great  extent  lost,  but  the 
oxidation  of  hypoxanthine  to  uric  acid  was  a  different  matter, 
and  was  not  affected  by  removal  of  the  liver.  Attempts  to 
1  v.  Mach,  S.  A.  24,  398,  1888. 


184       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

prove  the  direct  conversion  of  xanthine  bases  into  uric  acid  in 
mammalian  animals  and  man,  however,  were  for  some  time 
unsuccessful.  Horbaczewski  in  1891  found  that  food  containing 
nucleine  increased  the  uric  acid  excretion  in  rabbits  and  men ; 
but  he  explains  this  by  supposing  that  it  was  the  leucocytosis 
set  up  by  the  nucleine  and  not  the  nucleine  itself,  that  gave  rise  to 
the  uric  acid ;  others  subsequently  confirmed  his  result  again 
and  again,  but  showed  that  his  explanation  would  not  hold.1 
But  Minkowski  proved  that  even  in  dogs,  though  much  less 
than  in  men,  feeding  with  nucleic  acid  from  the  testes  of  the 
salmon  caused  a  rise  in  the  excretion  of  uric  acid,  and  that  the 
xanthine  bases  themselves  too,  especially  hypoxanthine,  had  the 
same  effect.2  The  old  idea  that  uric  acid  was  formed  in  the 
catabolism  of  proteids  in  general,  but  was  for  the  most  part 
normally  converted  into  urea,  and  that  excess  of  uric  acid  could 
be  accounted  for  by  failure  of  this  final  stage  of  nitrogenous 
metabolism,  had  to  be  abandoned ;  for  proteids  such  as  those  in 
eggs,  which  contain  no  purine,  do  not  increase  the  uric  acid 
excretion.3  The  mistake  had  been  fostered  by  the  fact  that 
meat  contains  purine  bases,  though  but  little  nucleine  :  100  g.  of 
meat  has  on  an  average  60  mg.  of  purine  bases  in  it,  only  one 
quarter  of  which  is  combined.4 

Incidentally  it  should  be  noted  that  the  uric  acid  excretion 
was  found  to  be  less  than  the  theoretical  yield  for  a  given 
amount  of  purine,  in  man  as  well  as  in  the  dog ;  but  in  this 
animal  the  loss  of  purine  in  the  body  was  much  greater  than  in 
man. 

But  the  formation  of  uric  acid  from  nucleic  acid  and 
purine  contained  in  the  food,  does  not  account  for  all  the  uric 
acid  in  the  urine,  since  it  is  well  known  that  the  urine  contains 
uric  acid  when  no  purine  of  any  kind  is  allowed  to  enter  the 
body.  The  source  that  suggests  itself  for  this  endogenous 

1  Horbaczewski,  M.  f.  Ch.  12,  221  ;  Weintraud,  B.  k.   W.^  1895  ;  Hess 
and  Schmoll,  S.  A.  37,  243,  1896. 

2  Minkowski,  S.  A.  41,  375,  1898. 

3  Hess  and  Schmoll,  loc.  cit. 

4  Burian  and  Schur,  Pfl.  A.  80,  241,  1900. 


vni.]  ENDOGENOUS  URIC  ACID  185 

purine  is,  naturally  enough,  the  nucleic  acid  of  the  cells  in  the 
body.  The  chemical  changes  would  be  the  same  in  the  one 
case  as  in  the  other.  But  it  is  an  assumption  that  is  not 
necessarily  justifiable,  to  suppose  that  nuclear  substance, 
wherever  it  may  be  in  the  body,  when  breaking  down,  breaks 
down  in  this  particular  way  merely  because  the  reactions 
involved  are  not  altogether  foreign  to  animal  organisms.  And 
methods  have  not  yet  been  discovered  by  which  the  secrets  of 
nuclear  metabolism  can  be  revealed. 

Such  evidence  as  we  have,  however,  is  strongly  in  favour 
of  this  hypothesis,  which  is,  in  fact,  very  generally  adopted. 
We  know  that  in  autolysis  nucleic  acids  give  up  purine  bases.1 
We  know  that  while  the  bases  actually  present  in  nucleic  acids 
and  set  free  by  hydrolysis  with  acids  are  the  amino  bases 
adenine  and  guanine  (6-amino  purine  and  2-amino  6-oxypurine 
respectively),2  the  bases  that  are  found  in  the  solution  of  the 
organs  that  results  from  their  autolysis  are  much  rather  the 
oxypurines  corresponding  to  these,  hypoxanthine  and  xanthine. 
The  amino  purines  tend  to  disappear  and  give  place  to  the 
oxypurines  during  autolysis,3  and  it  often  happens  that  no 
trace  of  either  adenine  or  guanine  can  be  detected  in  the  final 
products.  Also  if  these  bases  are  added  to  an  extract  of  the 
organs,  under  these  circumstances  too  they  are  oxidised.  In 
some  cases  the  reaction  is  confined  to  adenine,  and  guanine  is 
unaffected,  in  others  both  alike  are  thus  denitrified  and  oxidised.4 
These  autolytic  reactions,  which  are  ascribed  to  enzymes  known 
as  adenase  and  guanase,  bring  us  nearer  to  uric  acid.  But  on 
referring  to  the  molecular  diagrams  of  these  substances  it  will 
be  obvious  that  a  second  reaction,  different  from  this,  is  neces- 
sary in  order  to  produce  uric  acid.  The  group  in  adenine  or 

1  Salkovvski,     Z.    f.     k.    M.,     1890;     cf.     Salomon,    M.    /.,    p.    106, 
1881. 

2  Schmiedeberg,  S.  A.  43,  57,  1900  ;  Kossel  and  Neumann,  B.  27,  2215, 
1894;  Osborne  and  Harris,  H.-S.  Z.  36,  102,  1902;  Levene,  H.-S.  Z.  32, 
545,  1901  ;  and  37,  404,  1902.     Cf.  Burian,  Ergeb.  iii.,  p.  86. 

3  Jones,  H.-S.  Z.  42,  343,  1904. 

4  Jones,  H.-S.  Z.  45,  89,  1905. 


186       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LKCT. 

guanine  which  is  affected  by  the  change  we  have  so  far  con- 

,\  \ 

sidered  is  C  =  NH,  and  this   is  converted   into  C  =  O,     But  to 

I  I 

convert  xanthine  into  uric  acid  or  hypoxanthine  into  xanthine 
the  change  is  a  different  one,  and  involves  the  oxidation  of 
the  group 

— N  — NH 

\  \ 

C  .  H          to  the  group  C=O 

The  formation  of  uric  acid  from  purine  bases  resulting  from 
this  change  was  first  observed  by  Horbaczewski,  in  his  well- 
known  experiments  with  the  spleen,  which  marked  an  epoch 
in  the  physiology  of  uric  acid.1  Spleen  pulp  was  heated  with 
eight  to  ten  volumes  of  water  at  50°  C.  for  some  hours  till 
decomposition  began.  It  was  then  precipitated  with  basic  lead 
acetate  and  filtered ;  and  the  filtrate  mixed  with  an  equal 
volume  of  blood  was  kept  at  38°  C.  with  a  current  of  air  passing 
through  it.  After  a  few  hours  adenine  and  guanine  could  not 
be  found,  and  uric  acid  had  been  formed.  He  thought  that 
the  initial  bacterial  decomposition  was  necessary,  but  that  at 
a  certain  stage  it  must  be  checked  by  the  use  of  lead  acetate. 
But  it  was  proved  that  in  that  he  was  mistaken.  Spitzer 2  found 
that  extracts  of  the  spleen  and  liver  treated  with  thymol 
chloroform  or  sodium  fluoride  converted  the  xanthine  and 
hypoxanthine  which  he  added  to  them,  when  supplied  with  a 
current  of  air  at  50°  C.,  into  uric  acid.  And  Schittenhelm 3  was 
able  to  precipitate  with  two  volumes  of  saturated  ammonium 
sulphate  solution  something  contained  in  the  extract  of  the 
liver,  spleen,  or  lungs,  a  solution  of  which  in  a  current  of  air 
converted  guanine  in  some  cases  quantitatively  into  uric  acid. 
If  the  air-stream  were  omitted,  xanthine  was  formed,  and  not 

1  Horbaczewski,  M.f.  Ch.  12,221,  1891. 

2  Spitzer,  Pfl.  A.  76,  192,  1899. 

3  Schittenhelm,  ff.-S.  Z.  42,  253,  and  43,  228,  1904.     Cf.,  too,  Burian, 
H.-S.  Z.  43,  497,  1904. 


viii.J          SYNTHESIS  OF  URIC  ACID  IN  THE  BODY  187 

uric  acid ;  in  other  words  the  reaction  involving  denitrification 
took  place,  but  not  the  second  reaction  by  which  the  CH  group 

is  oxidised. 

In  this  way  we  have  learnt  to  look  upon  the  formation  of 
uric  acid  from  nuclear  purine  as  a  probable  reaction  in  nuclear 
metabolism.  But  it  is  not  proved  in  the  same  way  that  the 
formation  of  uric  acid  from  food  purine  is  proved.  And  in 
order  to  account  for  the  uric  acid  that  is  excreted  when  no 
purine  is  taken  with  the  food,  we  are  not  compelled  to  take 
this  probability,  however  great  it  may  be,  for  proof.  For  there 
is  another  way  in  which  it  is  possible  that  such  endogenous 
uric  acid  may  arise,  and  that  is  by  synthesis  from  material  that 
is  not  already  in  the  purine  form.  It  has  long  been  known 
that  birds  and  reptiles  excrete  70  per  cent,  of  their  nitrogen 
as  uric  acid.  This  amount  cannot  all  have  been  purine  to 
start  with,  and  in  fact  all  kinds  of  nitrogenous  compounds  in 
these  animals  contribute  to  the  output  of  uric  acid.  Minkowski's 
experiments  on  the  removal  of  the  liver  in  geese  made  it 
probable  that  in  this  synthesis  ammonia  is  first  converted  into 
urea,  and  the  urea  instead  of  being  ejected  at  once  is  further 
elaborated  by  condensation  with  lactic  acid  to  uric  acid.  This 
conception  was  confirmed  by  Salaskin  and  Kovalevski,1  who 
on  perfusion  of  the  excised  livers  of  birds  with  blood  containing 
ammonium  lactate  found  that  uric  acid  was  formed.  And 
Wiener2  proved  it  in  another  way.  Since  urea  administered 
to  birds  is  excreted  as  uric  acid,  whatever  else  is  necessary  for 
'the  synthesis  of  uric  acid  from  urea  must  be  present  in  the 
organism  in  larger  amount  than  is  required  for  the  ordinary 
output  of  uric  acid.  But  if  the  dose  of  urea  be  progressively 
increased,  a  point  is  reached  at  which  the  animal  begins  to 
excrete  the  urea  unchanged,  at  which,  it  may  be  assumed,  the 
supply  of  the  other  factor  necessary  for  the  synthesis  is  exhausted. 
If  at  this  point  lactic  acid  be  injected  in  addition  to  the  urea, 
then  the  urea  is  no  longer  excreted  unchanged,  but  uric 

1  Salaskin  and  Kovalevski,  H.-S.  Z.  33,  210,  1901. 

2  Wiener,  H.  £.,  ii.,  42,  1902. 


188        THE  METABOLISM  OF  CYCLIC  FORMATIONS     [I.ECT. 

acid  begins  to  be  formed  again.  Still  more  effectual  than 
lactic  acid  in  this  respect  are  certain  other  organic  acids,  also 
containing  three  carbon  atoms  in  chain,  notably  malonic  acid, 
COOH.CH2.COOH,  and  tartronic  acid,  COOH.CHOH.COOH. 
These  are  substances  which  might  well  be  formed  from  lactic 
acid  by  oxidation  ;  and  the  reason  why  they  give  better  yields 
of  uric  acid  than  does  lactic  acid  itself  may  well  be  that  lactic 
acid  does  not  all  of  it  undergo  oxidation  into  these  substances, 
and  that  part  of  it,  reacting  in  other  ways,  is  diverted  into  other 
channels,  and  so  rendered  unavailable  for  the  synthesis.  In 
his  experiments,  malonic,  tartronic,  and  mesoxalic  acids  gave 
quantitative  yields:  glycerine,  lactic  and  pyruvic  acids  about 
40  per  cent. ;  while  the  four  carbon  acids,  butyric,  succinic,  and 
malic,  gave  none. 

With  the  clearer  insight  into  the  mode  of  origin  of  uric 
acid  in  birds  thus  obtained,  the  question  arose  whether  the 
reactions  involved  were  peculiar  to  those  animals  whose  nitrogen 
excretion  is  mainly  in  this  form.  Wiener  tested  some  of  these 
acids  on  human  beings,  and  found  a  definite,  though  small, 
increase  in  the  uric  acid  excretion  after  giving  malonic,  lactic 
or  dialuric  acid  (the  monoureide  of  tartronic  acid, 

/NH .  CO. 

C0\  >CHOH)  ; 

\NH .  CO/ 

and  finally  he  found  that  an  extract  of  ox-liver,  containing 
0.2  per  cent,  sodium  fluoride,  after  being  shaken  with  tartronic 
acid  for  four  hours,  contained  more  uric  acid  than  a  portion 
similarly  treated  but  with  no  addition.  '  These  experiments  led 
him  to  the  view  that  a  synthesis  of  uric  acid  from  urea  and 
oxidation  products  of  lactic  acid  occurred  in  the  liver  of 
mammalian  animals  as  in  that  of  birds,  though,  of  course,  to  a 
very  much  smaller  extent. 

Wiener  was  led  to  making  these  experiments  by  some 
curious  results  noted  by  Hopkins  and  Hope.1  They  found  that 
when  they  took  sweetbread  which  had  been  digested  artificially 

1  Hopkins  and  Hope,//,  of  Phys.  23,  271,  1898. 


viii.]  SYNTHESIS  OF  HYPOXANTHINE  189 

with  pepsine,  and  then  freed  by  filtration  from  nucleine,  a  marked 
increase  in  the  uric  acid  excreted  was  constantly  observed  in  spite 
of  the  removal  of  the  nucleine.  But  in  these  experiments  it  was 
not  quite  clear  that  there  were  not  sufficient  purine  bases  free  in 
the  filtered  solution  of  digested  thymus  to  account  for  the 
increased  uric  acid.  And  Wiener's  experiments,  though  sugges- 
tive, are  not  universally  accepted  as  establishing  the  synthesis 
which  he  supposes. 

Burian  is  also  one  of  those  who  are  not  satisfied  that  the 
nuclear  metabolism  of  the  body  should  be  made  to  account  for 
the  whole  of  the  endogenous  uric  acid.1  But  he  has  been  led  to 
a  conception  of  the  synthesis  of  uric  acid  which  is  entirely 
different  from  that  propounded  by  Wiener.  Endogenous  uric 
acid  takes  its  origin,  according  to  him,  principally  in  the  muscles. 
Here  in  the  first  instance  hypoxanthine  is  formed  by  synthesis, 
and  this  is  then  subsequently  oxidised  to  uric  acid  by  the 
ferment  effecting  this  change  which  the  muscles  are  known  to 
contain.  This  view  is  based  on  the  fact  determined  by  him,  that 
muscular  activity  is  followed  in  the  first  hour  by  increased 
excretion  of  purine  bases  other  than  uric  acid,  and  then  for  an 
hour  or  two  more  by  increased  excretion  of  uric  acid.2  And  in 
confirmation  of  this  he  finds  that  excised  muscles  of  dogs,  per- 
fused with  blood,  give  up  to  the  blood  during  activity,  at  first 
more  bases  and  afterwards  more  uric  acid.  He  thinks,  then,  that 
the  muscles  are  the  principal  source  of  endogenous  uric  acid,  that 
in  producing  it  they  build  up  the  purine  ring,  and  that  hypoxan- 
thine is  a  product  of  muscular  metabolism,  just  as  kreatine  is  a 
substance  which  there  is  also  reason  for  thinking  is  produced  in 
quantities  that  vary  with  muscular  activity.  How  the  hypoxan- 
thine and  kreatine  are  put  together  is  not  yet  known,  but  it  is  of 
interest  in  this  connection  to  remember  that  kreatinine  is  essen- 
tially an  imidazol  derivative,  that  the  imidazol  formation  occurs 
in  purine  derivatives,  and  that  methyl  imidazol  can  be  obtained 
in  vitro  directly  from  sugar  and  ammonia  by  the  action  of  sun- 
light. There  is  not  a  particle  of  evidence  that  there  is  any 

1  Burian,  H.-S.  Z.  43,  533,  1905. 

2  Cf.,  too,  van  Hoogenhuyze  and  Verploegh,  ff.-S.  Z.  46,  442,  1905. 


190       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

genetic  relationship  of  this  kind  between  these  substances ;  but 
there  is  not  a  particle  of  evidence  for  any  theory  to  account  for 
their  origin  in  the  muscles,  either  independently  or  in  association. 

Then,  lastly,  in  addition  to  the  three  modes  in  which  it  has 
been  supposed  that  uric  acid  may  arise  in  the  body,  two  of  which 
are  by  oxidation  of  ready-made  purine,  and  the  third  by  some 
synthetic  process,  we  have  to  remember  that  uric  acid  itself  is 
not  indestructible. 

Nearly  sixty  years  ago,  Wohler  and  Frerichs l  showed  that 
uric  acid  given  to  dogs  was  converted  into  urea,  and  this  has 
been  confirmed  again  and  again  since  then.  Spiegelberg  found 
that  he  could  in  these  animals  recover,  as  uric  acid  in  the  urine, 
only  about  5  per  cent,  of  the  uric  acid  administered  by  the 
mouth  if  the  dogs  were  full-grown,  but  in  puppies  as  much  as 
50  per  cent.  Burian  and  Schur 2  determined  what  they  call  the 
integrative  factor  for  uric  acid  ;  that  is  to  say,  the  number  of 
milligrams  of  uric  acid  that  must  reach  the  blood  in  order  that 
i  milligram  should  be  excreted  in  the  urine :  in  dogs  this 
integrative  factor  is  22,  agreeing  closely  with  Spiegelberg's 
figure,  in  rabbits  6,  in  man  2.  This  factor  is  clearly  a  measure 
of  the  destruction  of  uric  acid  in  the  organism  of  the  species ; 
and  they  argued  that  they  could  determine  the  amount  of  uric 
acid  that  had  been  thrown  into  the  circulation,  whether  exo- 
genous or  endogenous,  by  multiplying  the  amount  excreted  by 
the  factor  for  the  species.  Whether  such  calculations  are  con- 
vincing or  not,  they  showed  that  the  destruction  of  uric  acid  is 
carried  out  largely  in  the  liver.  For  excision  of  the  kidneys 
caused  no  accumulation  of  uric  acid  in  the  blood  so  long  as  it 
was  allowed  to  circulate  through  the  liver ;  but  if  the  liver  was 
cut  out  of  the  circulation,  in  addition  to  the  kidneys  being 
removed,  then  accumulation  of  uric  acid  did  take  place.  And 
there  seems  to  be  no  doubt  whatever  that  besides  the  liver  other 
organs  are  also  able  to  destroy  uric  acid.  Wiener 3  found  that 

1  Wohler  and  Frerichs,  Ann.  65,  1848. 

2  Burian  and  Schur,  Pfl.  A.  80,  241,  1900 ;  and  87,  239,  1901. 

3  Wiener,  S.  A.  42,  375,  1899.    Cf.,  too,  Jacoby,   V.  A.  157,  235,  1899; 
Lang,  H.  #.,  v.,  330,  1904. 


viii.]       DESTRUCTION  OF  URIC  ACID  IN  THE  BODY       191 

extracts  of  the  liver  of  dogs  and  pigs,  and  of  the  kidney,  though 
not  the  liver,  of  oxen,  shaken  for  four  hours  at  body-temperature 
with  uric  acid,  contained  at  the  end  of  that  time  a  much  smaller 
amount  of  uric  acid  than  portions  that  had  been  boiled,  but 
otherwise  treated  in  the  same  way.  In  some  cases,  out  of  140  mg. 
of  uric  acid  added  to  each  sample  only  3  or  4  mg.  could  be 
recovered,  while  in  the  boiled  sample  nearly  the  whole  was 
recovered.  Schittenhelm 1  has  been  able  by  means  of  uranyl 
acetate  to  separate  the  substance  in  the  kidney  to  which  this 
action  is  due,  and  found  that  a  solution  of  it,  supplied  with  air  at 
38°C.  in  the  presence  of  chloroform,  destroyed  from  80  to  100 
per  cent,  of  the  uric  acid  that  he  added,  whereas  if  the  solution 
were  boiled  this  was  not  the  case. 

But  what  it  is  that  is  formed  from  the  uric  acid  which  dis- 
appears in  the  body,  or  in  these  experiments  in  vitro,  has  not 
been  determined.  The  decomposition  of  uric  acid  under  almost 
every  possible  variety  of  conditions  was  studied,  in  the  early 
attempts  to  determine  its  constitution.  And  many  different 
substances  were  obtained  from  it.  When  heated  with  hydro- 
chloric acid  to  I7O°C.  it  breaks  up  into  glycocoll,  carbonic  acid, 
and  ammonia.  It  was  accordingly  surmised  that  glycocoll  might 
be  formed  when  uric  acid  was  destroyed  in  the  body.  Wiener 2 
tested  this  conjecture  by  saturating  rabbits  with  benzoic  acid 
introduced  into  the  stomach,  giving  quantities  larger  than  the 
animal's  own  stock  of  glycocoll  could  combine  with  to  form 
hippuric  acid.  If  uric  acid  were  then  injected  subcutaneously, 
more  hippuric  acid  was  formed,  and  at  the  same  time  the  amount 
of  benzoic  acid  that  could  be  given  without  killing  the  animal 
was  increased.  He  also  found  that  when  an  extract  of  ox- 
kidney,  obtained  by  shaking  the  pulped  organ  with  normal 
saline  containing  0.2  per  cent,  of  sodium  fluoride  for  some  time 
and  then  straining,  was  digested  with  uric  acid,  not  only  did  the 
uric  acid  disappear,  as  we  have  seen  above,  but  glycocoll  was 
found  in  larger  quantities  than  was  the  case  if  no  uric  acid  was 
added.  The  amount  of  glycocoll  actually  found  was  enough  to 

1  Schittenhelm,  H.-S.  Z.  45,  160,  1905. 

2  Wiener,  S.  A.  40,  313,  1898. 


192        THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

account  for  about  half  the  amount  of  uric  acid  that  had 
disappeared,  on  the  supposition  that  a  molecule  of  uric  acid  gives 
rise  to  one  of  glycocoll.  It  is  possible  that  the  glycocoll  is 
not  formed  directly  from  the  uric  acid ;  some  intermediate 
product  may  intervene  in  a  reaction  taking  place  in  two 
stages,  and  the  amount  of  glycocoll  found  in  this  experiment 
may  have  been  low,  for  the  reason  that  some  of  the  uric  acid 
had  been  carried  through  the  first  stage  but  not  the 
second. 

But  if  it  is  proved  that  uric  acid  in  the  kidney  gives  rise  to 
glycocoll,  it  does  not  follow  that  uric  acid  is  disposed  of  in  the 
same  way  in  other  organs  of  the  body  in  which  it  is  known  to  be 
destroyed.  There  are  other  destructive  decompositions  of  uric 
acid  that  have  been  well  known  from  the  early  days  of  the 
chemistry  of  this  substance.  With  permanganate  of  potash  in 
the  cold,  uric  acid  is  oxidised  to  allantoine — 

NH     .     CO  NH2 

CO  C— NHX  >      CO        .  CO.NH, 

\  II         >co  \  >co 

NH     .     C— NH/  NH.CH.NH/ 

Now  allantoine  is  found  in  the  urine  of  calves  and  cows,1  in 
that  of  new-born  children,2  and  not  infrequently  in  that  of  dogs 
and  even  adult  human  beings.3  On  autolysis  of  the  liver 
allantoine  is  formed,  and  in  hydrazine  poisoning,  in  which  the 
liver  undergoes  severe  disintegrative  change,  this  organ  is  found 
to  contain  much  allantoine.4  Salkowski  administered  8  g.  of 
uric  acid  to  a  dog,  and  found  that  the  urine  contained  about  1.5  g. 
of  allantoine ;  and  he  notes  that  dogs  fed  on  meat  frequently 
excrete  allantoine,  but  show  in  this  respect  individual  peculiari- 
ties, the  allantoine  excreted  in  some  animals  being  replaced  by 

1  Salkowski,  H.-S.  Z.  42,  220,  1904. 

2  Prout,  Med.-Chir.  Trans.  8,  526,  1818. 

3  Cf.  Minkowski,  S.  A.  41,  397,  1898. 

4  Pohl,  S.  A.  48,  367,  1903. 


viii.]  ALLANTOINE  AND  OXALIC  ACID  193 

uric  acid  in  others.1  Minkowski  found  that  hypoxanthine 
administered  to  dogs  appeared  as  allantoine  in  the  urine,  while 
in  man  it  was  excreted  as  uric  acid.  After  feeding  on  thymus, 
too,  dogs  excreted  considerable  amounts  of  allantoine,  while  in 
men  under  these  conditions  none  was  excreted.  All  this 
suggests  that  allantoine  is  formed  when  uric  acid  is  destroyed 
in  the  body,  and  that  allantoine  is  not  commonly  found  in  the 
urine  of  either  dogs  or  men,  because  it  is  itself  further  acted  on 
and  destroyed.  Given  by  the  mouth,  it  is  true,  it  is  not  broken 
up  in  the  dog  ;  as  much  as  90  per  cent,  has  been  recovered 
unchanged  in  the  urine.2  But  it  may  well  be  that,  when  it  arises 
from  uric  acid  in  the  cells,  it  is  attacked  on  the  spot,  and  not 
allowed  to  reach  the  blood  or  the  kidneys.  In  man,  on  the 
other  hand,  even  when  taken  by  the  mouth,  from  50  to  70  per 
cent,  of  it  disappears,  and  only  the  smaller  part  is  found  in  the 
urine. 

Then,  again,  from  allantoine  in  vitro  oxidising  agents  give 
rise  to  the  formation  of  oxalic  acid  and  urea.  Even  potash 
splits  off  oxalic  acid  from  this  body.3  So,  too,  the  oxidation 
of  uric  acid  itself  with  nitric  acid  finally  results  in  the 
production  of  oxalic  acid.  And  oxalic  acid  occurs  constantly 
in  small  quantity  in  the  urine,  occasionally  in  larger  amount 
as  a  result  of  disordered  metabolism,  in  diabetes,  or  in 
chronic  gastric  disturbances.4  Now  this  oxalic  acid  is  very 
often  in  part  derived  from  ingested  oxalic  acid,  for  it  has 
been  proved  that  this  substance,  not  only  when  absorbed  from 
the  stomach,  but  also  when  small  quantities  are  injected  sub- 
cutaneously,  is  excreted,  in  the  latter  case  quantitatively,  in  the 
urine.5  But  in  addition  to  the  varying  amounts  that  are 
introduced  into  the  system  with  vegetable  foods,  this  acid  is 

1  Salkowski,  B.  7,  719, 1876  ;  and  11,  501,  1878  ;  cf.,  too,  H.-S.  Z.  35,  493, 
1902. 

2  Poduschka,  S.A.  44,  64,  1900;  cf.,  too,  Luzzatto,  ff.-S.  Z.  38,  537,  1903. 

3  Ad.  Claus,  B.  7,  226,  1874. 

4  Lewin,  H.  B.  I,  490  ;  Baldwin,  M.J.  715,  1900. 

5  Pierallini,  M.  /.  714,  1900  ;  Pohl,  S.  A.  37,  413,  1896 ;  Faust,  S.  A.  44, 
235,  1900. 

N 


194       THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

a  product  of  the  metabolism  of  the  body,  and  the  fact  that 
whereas  casein  does  not  increase  the  output,  meat  and  still 
more  thymus  does,  led  Salkowski  to  suspect  that  it  was  derived 
from  nucleine.1  This  may  mean  that  it  is  formed  in  the 
destruction  of  uric  acid,  and  be  one  of  the  substances  that  we 
are  searching  for.  It  has  been,  as  a  matter  of  fact,  found  that 
allantoine  in  rabbits  gives  rise  to  a  very  definite  degree  of 
oxaluria ;  but,  on  the  other  hand,  neither  in  dogs  nor  in  rabbits 
is  there  any  more  oxalic  acid  excreted  as  a  result  of  adding 
uric  acid  to  the  food.2 

It  is  not  possible,  therefore,  to  say  for  certain  what  becomes 
of  the  uric  acid  that  is  disposed  of  in  the  body  before  it  can 
reach  the  urine.  Glycocoll,  allantoine,  and  oxalic  acid  may  each 
of  them,  in  certain  cases  at  any  rate,  account  for  some  of  it. 
But  there  is  no  doubt  that  uric  acid  is  not  necessarily  a  final 
product  that  undergoes  no  further  change.  The  purine  ring  is 
not  indestructible,  and  this  fact  has"  to  be  taken  into  account 
in  pathology  as  well  as  in  physiology.  And  if  there  is  doubt 
about  the  last  stages  in  the  metabolic  processes  leading  to  the 
destruction  of  purine,  the  earlier  stages  are  intelligible,  and 
probably  throw  light  on  the  fate  of  ring  structures  in  general 
in  the  body.  The  forms  of  purine  which  actually  occur  in  the 
nucleic  acids  are  the  amino  derivatives.  By  the  gradual  in- 
sinuation of  oxygen  atoms  wherever  possible,  leading  to  the 
formation  of  uric  acid,  the  ring  is  so  weakened  that  unless 
excreted  at  this  stage  it  breaks  up.  Till  we  know  more  about 
the  origin  of  kreatine  in  the  muscles,  it  may  be  fanciful  to  draw 
a  parallel  in  the  case  of  this  substance.  But  it  may  be  that  in 
kreatinine  we  have  the  last  stage  before  the  disappearance  of  an 
imidazol  ring. 

In  this  connection  we  are  led  finally  to  what  is  known  of 
the  fate  of  the  aromatic  ring  in  tyrosine  and  phenyl-alanine. 
Our  conceptions  on  this  point  have  been  made  clearer  by  the 
study  of  the  disorder  known  as  alcaptonuria. 

1  Salkowski,   B.  k.    W.  20,  1900;   Luthje,  M.  /.  584,    1898;   Lommel, 
M-  /336,  1899. 

2  Luzzatto,  n.-S.  Z.  37,  228  ;  and,  38,  542. 


viii.]  ALCAPTONURIA  195 

In  this  interesting  anomaly  of  metabolism  the  urine  contains 
considerable  amounts  of  homogentisic  acid ;  that  is  to  say,  1.4 
dioxybenzene  acetic  acid,1 

•0- 

CH2 .  COOH 

Baumann,  who  first  discovered  the  presence  of  this  abnormal 
substance  in  the  urine,  showed  that  the  administration  of  tyrosine 
to  persons  who  had  alcaptonuria  caused  an  increase  in  the 
amount  of  homogentisic  acid  in  the  urine.  But  he  thought  that 
the  homogentisic  acid  was  formed  by  some  peculiar  variety  of 
bacteria  in  the  intestine.  It  has  since  then  been  shown  that 
the  homogentisic  acid  bears  a  constant  proportion  to  the 
nitrogen  excreted,  whatever  the  amount  of  proteid  taken  may 
be,  and  even  when  proteid  is  almost  excluded  from  the  diet. 
Since  the  nitrogen  excreted  under  such  circumstances  comes 
from  the  internal  metabolism  of  the  body,  it  is  difficult  to  ascribe 
a  different  source  to  the  homogentisic  acid,  and  Baumann's 
idea  that  it  is  not  a  product  of  metabolism  at  all  has  been 
abandoned.2  From  the  study  of  the  ratio  of  the  homogentisic 
acid  to  nitrogen  excreted  another  point  was  made  put,  that 
this  ratio  was  higher  than  could  be  accounted  for,  even  on  the 
supposition  that  all  the  tyrosine  set  free  in  proteid  metabolism 
underwent  this  change.3  It  was  then  found  that  phenyl-alanine 
behaved  like  tyrosine  in  persons  with  this  affection,  and  was 
almost  entirely  converted  into  homogentisic  acid.4  This  makes 

1  Wolkow  and  Baumann,  H.-S.  Z.  15,  228,  1891. 

2  Falta,  B.  Cbl  3,  173,  1904  ;  cf.  Embden,  H.-S.  Z.  18,  304. 

3  Langstein  andE.  Meyer,  D.  A.f.  k.  M.  78  ;  and,  D.  R.  A.,  p.  383,  1903. 

4  Falta  and  Langstein,  H.-S.  Z.  37,  513,  1903. — It  is  well  known  that 
many  fungi  contain  a  "  tyrosinase  "  which  oxidises  tyrosine  to  a  dark  brown 
or  black  substance.     It  is  said  that  homogentisic  acid  is  formed.     Raper 
and  I  tried  to  see  if  these  fungi  oxidised  phenyl-alanine  similarly  ;  we  collected 
four  varieties  of  Russula  that  contain  tyrosinase,  and  found  that  while  the 
juice   oxidised  and  blackened  tyrosine,  it  had  no   effect  apparently  upon 
phenyl-alanine. 


196      THE  METABOLISM  OF  CYCLIC  FORMATIONS     [LECT. 

it  somewhat  easier  to  understand  the  conversion  of  tyrosine  in 
which  the  hydroxyl  group  is  in  the  para  position  to  the  side 
chain,  into  a  substance  in  which  two  hydroxyl  groups  are  in 
the  1-4  positions,  the  one  ortho  and  the  other  meta  to  the 
side  chain.  If  the  tyrosine  be  reduced  to  phenyl-alanine  it 
would  still  be  converted  into  the  same  product  in  these  persons. 
Further,  it  has  been  shown  that  phenyl-lactic  acid  and  phenyl- 
pyruvic  acid  also  give  rise  to  homogentisic  acid,  while  phenyl- 
acetic  acid  does  not,1  so  that  it  has  been  suggested  that  the 
sequence  of  events  in  the  metabolism  of  tyrosine  under  these 
conditions  is  to  be  represented  by  the  following  formulae  : 2 — 

OH 


CH2.C 


Tyrosine. 


H 

.  COOH  CH, .  C<"         .  COOH 

NHo 


Phenyl-alanine. 

HO 


.H 


CH  .  C 


OH 


.  COOH      CH  .  CO  .  COOH       CH  .  COOH 


Phenyl-lactic  acid.  Phenyl-pyruvic  acid.  Homogentisic  acid. 

Now  the  physiological  importance  of  all  this  is  derived  from 

1  Falta,  M.  M.  W.,  p.  1846,  1903  ;  Embden,  H.-S.  Z.  18,  316. 

2  The  occasional  occurrence  of  uroleucic  acid, 


HO 


/OH 


JI 
OH 


.COOH 


in  alcaptonuric  urine  would  imply  that  the  oxidation  of  the  ring  would  occur 
at  an  earlier  stage  than  indicated  in  this  scheme. 


vin.]  TYROSINE  IN  METABOLISM  197 

the   fact   that   in    normal  men  the   aromatic   ring   In   gentisic 
acid 

HO 


OH 
COOH 

taken  by  the  mouth  disappears  almost  entirely,  just  as  in 
tyrosine  and  phenyl-alanine  it  does  ;  but  in  alcaptonuric  patients 
it  is,  like  the  homologous  acid,  excreted  quantitatively  unchanged.1 
It  is  argued,  therefore,  that  normally  these  phenyl  derivatives, 
tyrosine  and  phenyl-alanine,  are  oxidised  to  homogentisic  acid 
just  as  they  are  in  alcaptonuria,  but  at  this  point  the  difference 
between  the  normal  and  the  abnormal  metabolism  shows  itself; 
normally  the  introduction  of  the  two  hydroxyl  groups  into  the 
benzene  ring  is  the  signal  for  the  destruction  of  the  ring.  But 
in  the  alcaptonuric  individual  the  ring  for  some  reason  will 
not  split :  this  is  the  reaction  that  fails.2 

It  will  be  remembered  that  tyrosine  in  the  intestine  is  to  a 
varying  extent  converted  by  bacteria  in  oxyphenyl-propionic 
acid  and  the  corresponding  acetic  acid  compound,  both  of 
which  may  appear  in  the  urine  or  be  converted  into  cresol  or 
phenol  itself.  And  these  phenols  are  excreted  as  esters  of 
sulphuric  or  glycuronic  acid,  the  ring  having  escaped  without 
any  fundamental  change.  Now  it  has  been  found  that  in 
alcaptonuria  phenyl-propionic  and  phenyl-acetic  acid  are  not 
oxidised  to  homogentisic  acid.3  It  seems,  therefore,  that  the 
bacteria  denitrify  tyrosine  to  oxyphenyl-lactic  acid,  but  then 
reduce  this  to  phenyl-propionic  acid,  and  thereby  at  once  render 
the  ordinary  metabolic  changes  by  which  tyrosine,  including  the 
aromatic  ring,  is  completely  oxidised  in  the  body  no  longer 

1  Neubauer  and  Falta,  H.  S.  42,  84,  1904. 

2  It  is  interesting  to  note  that  a  disturbance  apparently  identical  with 
alcaptonuria    can   be   induced    in   plants,    metabolism    being  arrested  so 
that  homogentisic  acid  accumulates  in   the   roots.     Cf.  Czapek,   Ber.   der 
deutschen  Botan.  Ges.,  vols.  20  and  21,  1902  and  1903. 

3  Embden,  H.  S.  1 8,  316,  1893. 


198  THE  METABOLISM  OF  CYCLIC  FORMATIONS  [LECT.VIII. 

possible.  The  reduction  of  the  lactic  acid  group  to  propionic 
acid  does  not  prevent  the  oxidation  of  the  side  chain,  though 
this  probably  is  done  by  the  bacteria  too,  and  not  in  the  body  ; 
but  it  does  prevent  the  ring  from  being  broken  up.  It  appears 
that  the  normal  course  of  metabolism  is  for  the  lactic  acid  chain 
to  be  oxidised  to  an  acetic  acid  chain  by  way  of  pyruvic  acid, 

/H 

— CH9 .  C<    .  COOH  ->  — CH2 .  CO .  COOH  — >  — CH9 .  COOH 
X)H 

and  that  if  this  is  the  course  of  the  reaction  the  simultaneous 
oxidation  of  the  ring  to  hydroquinone  is  affected ;  while  if  the 
lactic  acid  is  first  reduced  to  propionic  acid,  this  oxidation  in 
the  ring,  which  in  normal  conditions  but  not  in  alcaptonuria 
leads  to  its  destruction,  is  no  longer  possible.  This  is  in  itself 
curious  and  interesting.  But  it  is  still  more  so  if,  as  appears 
probable,  it  applies  exactly  to  tryptophane  too.  We  know,  at 
any  rate,  that  bacteria  in  denitrifying  tryptophane  produce 
indol-propionic  acid,  not  indol-lactic  acid ;  that  the  indol- 
propionic  acid  is  further  converted  into  indol-acetic  acid,  scatol, 
and  indol  itself,  but  that  such  indol  groups  appear  in  the  form 
of  indoxyl  and  scatoxyl  esters  in  the  urine  with  the  ring  intact. 
We  know  at  the  same  time  that  tryptophane,  when  not 
appropriated  by  bacteria,  in  the  normal  course  of  metabolism 
disappears  entirely.  But  we  do  not  know  exactly  how  the 
reactions  leading  to  the  destruction  of  the  indol  ring  differ 
from  those  carried  out  by  bacteria  in  the  intestine.  But  the 
indol  ring  must  be  attacked  before  it  is  stripped  bare  of  its 
side  chain,  just  as  the  phenyl  group  must  in  tyrosine,  for  the 
naked  indol  ring  introduced  into  the  body  is  not  attacked. 


INDEX 


ABSORPTION  of  proteids,  rate  of,  137 
Acetic  acid  in  liver,  58 

metabolism,  103 
Acetic  aldehyde  in  synthesis  of  fat,  81 

from  lactic  acid,  53,  8 1 
Acetone  in  normal  expired  air,  1 10 
Acetonuria,  108 
Adamkievvicz,  reaction  of,  5 
Adenase,  185 
Adrenaline,  168 
Adsorption  and  catalysis,  18 
Alanine,  7 

as  source  of  sugar,  44 

compounds  of}  45,  179 
Albumins,  glucosamine  in,  32,  40 
Albumoses  in  blood,  133,  140 
Alcaptonuria,  195  seq. 
Alcohol  from  sugar  in  muscles,  66 
Alcoholic  fermentation,  14,  53 
Aldehydes,    formation    of,   from    oxy 

acids,  53 

Aldol  condensation,  21,  55,  82 
Alkalies,    action    of,    on     sugar,    25, 

5i 

Alkaloids  related  to  tryptophane,  1 76 
Allantoine  from  uric  acid,  192 

from  hypoxan thine,  193 
Amide  nitrogen  in  proteids,  152 
Amido  acids  as  source  of  sugar,  43  seq. 

formed  in  digestion,  127  seq. 

heat  equivalents  of,  128,  154 

and  nitrogenous  equilibrium,  130 

199 


Amido  acids  in  blood,  137  seq. 

in  tissues,  149 

in  metabolism,  148,  151  seq. 
Ammonia  in  acetonuria,  109 

in    blood     during     digestion,     140, 

I52. 
Ammonium  lactate  to  uric  acid  in  per- 

fusion  of  liver,  187 
Anti-catalysis  in  disease,  118 

in  health,  148 

and  antiseptics,  151 
Arabinose,  70 
Arginine,  u,  165 
Arginine  as  source  of  proline,  169 
Arsenious  acid  and  lactic  acid,  59 

and  fatty  liver,  118 
Aspartic  acid  as  source  of  sugar,  45 

in  peptic  digestion,  125 
Azelaic  acid,  104 

BAYER,  synthesis  of  carbohydrates  in 

plants,  9,  20 
Bial's  reaction,  33 
Biuret  reaction,  3 
Blood,  proteids  of,  141,  142 

rate  of  flow  of,  in  intestine,  137 
Blown  oils,  1 06 
Buchner,  zymase,  12,  52 
Butter  in  acetonuria,  161 
Butyric  fermentation,  53  seq. 
Butyric  acid  in  liver,  58 

in  metabolism,  103 


200 


INDEX 


CAPRONIC  ACID  from  lactic  acid,  80 

in  butyric  fermentation,  81 

in  metabolism,  103 

Carbohydrates,   synthesis    in    plants, 
9,20 

amount  of,  in  animal  body,  36 

digestion  of,  isothermic,  129 

effect  of,  in  acetonuria,  108 
Cartilage,  34,  43 
Casein  in  glycosuria,  42 

amide  nitrogen  in,  153 
Catalysis,  Ludwig,  13 

Ostwald's  definition,  16 

negative  in  disease,  118 
Cerebrines,  30 

Cetti,  metabolism  in  starvation,  97 
Choline,  168 
Chondrosine,  34 

Clupeine,  action  of  arginase  on,  166 
Co-ferments,  53 

Cohnheim  on  glycolysis  in  muscles,  66 
Cysteine,  conversion  into  tausine,  1  1 
Cystine  in  peptic  digestion,  125 

from  taurine,  n,  148 
Cytosine,  180 


D 


Denitrification   of   amido    acids,    15-1 
seq. 

of  purine  bases,  185 
Diabetes,  acetonuria  in,  108 

sugar  from  fat  in,  113 

Lepine's  theories,  65 
Diabetic    puncture    and    indicanuria, 

177 

Dialdane,  82 
Diainido  acids,  fate  of,  in  the  body, 

153 

Diamines  in  peptic  digestion,  125 
Digestion  of  proteids,  124  seq. 
Digestive  changes,  isothermic,  129 
Diphtheria  toxin  and  fatty  heart,  118, 

120 


Drechsel,  lysine,  10 
Drying  oils,  106 

EGGS,  metabolism  during  incubation, 

101 
synthesis  of  purine  in,  182 

Enzymes  and  vital  action,  12 
intracellular,  13,  57,  145 
physiological  classification,  13 

Erepsine,  126  seq. 

Erucic  acid  in  acetonuria,  1 1 1 

FARADAY,  catalysis,  19 

Fat,  amount  of,  in  animal  body,  35 

in  different  kinds  of  muscles,  99 

use  of,  in  muscular  activity,  100 

extraction  of,  120 

conversion     into    sugar,    112    sc?., 
119 

derived  from  proteids,  86,  164 
from  carbohydrates,  77,  85 

composition  of,  in  milk,  78 

source  of  energy  in  starvation,  98 

effect  on  acetonuria,  109 

iodine  value  of,  in  heart,  107 

compounds  of,  with  sugar,  31 
Fatty  acids,  in  metabolism,  103,  105 

stability  of,  106 

oxidation  of,  its  importance,  164 
Fatty  degeneration,  87  seq.,  i  \  7  seq. 
Fermentation,  14,  52  seq. 
Fever,  acetonuria  in,  108 

proteid  metabolism  in,  143 

kreatinine  excretion  in,  167 
Folin,  metabolism  on  diet  free  from 

proteids,  162 
Formic  acid  in  metabolism,  103,  105 

produced  by  yeast,  53 

from  lactic  acid,  81 
Formic  aldehyde  and  sugar  synthesis, 

in  plants,  10,  20,  48 

in  animals,  47,  164 

from  sugar,  180 
Fructose  converted  into  glucose,  25 


INDEX 


201 


GALACTOSE,  origin  of,  25 

in  fermentation,  55 

in  cerebrines,  31 

relation  to  arabinose,  71 
Globulin,  glucosamine  in,  32,  40 
Glucosamine,  constitution,  32 

estimation  of,  in  proteids,  32,  40 

as  source  of  glucose  and  glycogen, 

42 

Glucose,  conversion  into  lasvulose  and 
mannose,  25. 

conversion  into  imidazol,  179 
Glucoside  nature  of  proteids,  34 
Glutamic    acid    in    peptic    digestion, 
125 

relation  to  proline,  170 
Glyceric  acid  from  diamido  propionic, 

153 
Glyceric    aldehyde    as    precursor    of 

lactic  acid,  56 
Glycerine  as  source  of  sugar,  46 

origin  of,  95 
Glycoalbumoses,  34 
Glycocoll  as  source  of  sugar,  47 
in  urine  after  phosphorus,  139 
from  uric  acid,  191 
Glycogen,  amount  of,  in  animal  body, 

36,  114 

in  starvation,  38 
derived  from  glucose,  22 
from  other  sugars,  27 
from  proteids,  37 
from  glucosamine,  42 
from  alanine,  leucine,  44 
in  rigor  mortis,  59 
in  muscular  activity,  61,  62 
Glycolic  aldehyde,  47 
Glycolysis  in  blood,  65 
Glyco-proteids,  32,  40 
Glycosuria,  amount  of  sugar  excreted 

in,  36 

phlorrhizine,  36,  39 
pancreatic,  37-39,  45,  46 
v.  Diabetes. 


Glycuronic  acid  converted  into  xylose, 

29 
derived  from  glucose,  30 

from  proteids,  69 
in  urine,  30,  67 
Glyoxylic  acid  reaction,  5 
Guanase,  185 
Guanidine,  165 

derived  from  proteids,  167 

H^EMATINE,  123,  170 

Haematinic  acids,  171 

Haemoglobin,  122 

Heat,  effect  of,  on  reaction  velocity, 

16 
Heart,  fat  in,  99,  120 

iodine,  value  of,  107 
Heteroxanthine  from  xan thine,  168 
Hexone  bases  as  source  of  sugar,  44 
Hippuric  acid,  10 
from  phenyl    propionic    acid,   etc., 

105 

from  uric  acid,  191 
Histidine,  constitution,  179 
Homogentisic  acid,  195  seq. 
Hoppe-Seyler,  fatty  acids  from  lactic 

acid,  79 
Hybernation,  respiratory  quotient  in, 

112 
Hydrazine    poisoning,    allantoine    in, 

192 
Hydrogen  set  free  in  autolysis  of  liver, 

59 
Hydrolytic    changes    in    metabolism, 

150 
Hypoxanthine,   synthesis   in  muscles, 

189 

IMIDAZOL,  178,  179,  182 
Indicanuria,  176  seq. 
Indol,  172  seq. 
Intestine,  perfusion  of,  134 
synthesis  of  proteid  from  peptone 
in,  135,  141 


202 


INDEX 


Intestine,  fatty  acids  dissolved  in,  75 
Intracellular  enzymes,  13,  57 
Iodine  value  of  fat,  73,  89 

from  different  sources,  107 
lodo  fatty  acids,  73 
Isobutyric  acid  from  lactic  acid,  80 
Iso-cystine,  174 
Isomeric    transformations     of   sugar, 

25 
Iso-serine,  175 

JECORINE,  31 

KIDNEY,  fat  in,  99,  120 
of   ox,    uric    acid    destruction    in, 

191 
Kreatine    not    attacked  by   arginase, 

166 

origin  of,  194 
Kreatinine  excretion  on  different  diets, 

162 

endogenons,  166 
and  imidazol,  179 
Kiihne  on  proteid  digestion,  124 
Kynurenic  acid,  173 

LACTACIDASE,  52 

Lactic    acid    from    alanine,    44,    61, 

153 
from    sugars    in    fermentation,    51 

seq. 

in  the  blood,  65 
from  proteids,  60 
as  source  of  sugar,  45 
economises  proteid,  103 
in  muscle,  59,  61,  63 
in  liver,  58 

in  urine  of  birds  after  removal  of 
liver,  60 

in  fatigue,  57 
in  glycolysis  in  blood,  65 
in  synthesis  of  uric  acid,  187 
heat  equivalent  of,  63,  154 
Laevulose  converted  into  glucose,  25 


Lang,  denitrification,  15 1  seq. 
Laurie  acid  in  metabolism,  103 
Lebedeff's  experiment,  88 
Lecithine  in  jecorine,  31 
Le  Sueur,  decomposition  of  oxy-acids, 

104 

Leucine  as  source  of  sugar,  43 
in  peptic  digestion,  125 
heat  equivalent  of,  128 
Leucolytosis,  post-prandial,  134 

and  uric  acid,  184 
Linoleic  acid,  73,  78,  106 
Lipolysis,  74 
co-ferment  in,  53 
isothermic,  76,  129 
Liver,  acute  yellow,  atrophy  of,  139 
autolysis  of,  84 
fat,  synthesis  in,  85 
fatty  disease  in,  118 

change  in  phlorrhizine  glycosuria, 

119 
in   pancreatic    glycosuria,    115, 

119 

fat  from,  iodine  value,  107 
uric  acid,  synthesis  in,  188 

destruction  in,  191 
allantoine  formed  in,  192 
removal  of,  in  birds,  183 
oxidation  of  purine  bases  in,  186 
Lobry  de  Bruyn,  25 
Ludwig  on  catalysis,  13 
Lungs,  oxidation  of  purine  bases  in, 

1 86 
Liithje,  pancreatic  glycosuria,  37,  46 

MALONIC  ACID  in  synthesis  of  uric 

acid,  1 88 

Mannose,  conversion  into  glucose,  25 
Mannonic  acid,   action   of   pyrridine 

on,  27 

Margaric  acid,  78 
Meat,  purine  bases  in,  184 
Mesoporphyrine,  170 
Mesoxalic  acid,  188 


INDEX 


203 


Methylamine  derivatives,  168 
Methyl  mercaptan,  168 
Milk,  fats  in,  78 

synthesis    of    purine    bases    from, 
182 

Minkowski,  ~ ,  39 

N 

Molisch's  reaction,  33 

Moore's  test,  5 1 

Mucin,  32 

Muscles,  lactic  acid  in,  59,  61  seq. 

fat  in  different  varieties  of,  99 

glycolysis  in,  66 

Muscular  activity,  fat  used  in,  100 
kreatinine  excretion  in,  167 
hypoxanthine  formed  in,  189 
Myristic  acid  in  metabolism,  103 

NAPHTHALENE    sulphonyl    chloride, 

139,  148 

Nitrogen,  minimum  intake  of,  160 
cannot  be  stored,  157 
excretion  after  proteid  meal,  155 
not  a  measure  of  proteid   com- 
bustion, 155 

on  a  diet  free  from  proteids,  162 
Nitrogenous  equilibrium  on  diet  free 

from  proteids,  130 
Norisosaccharic  acid,  33 
Nucleic  acid,  pentose  in,  28 
glucoside  nature,  30 
pyrimidine  derivatives  in,  180 
in  autolysis,  185 

OILS,  "  drying,"  "blown,"  and  rancidity 

of,  1 06 
Oleic  acid,  constitution,  104 

in  acetonuria,  1 1 1 
Ostwald  on  catalysis,  16 
Oxalic  acid,  68 

origin  of,  193 

Oxamic  acid,  fate  of,  in  body,  153 
Oxidation   of   fatty  acids,    102,    105, 
no 


Oxy-acids,  decomposition  of,  104 

Oxy-aspartic  acid,  175 

Oxygen  consumption  in  starvation,  98 

on  various  diets,  100 

in  chick,  101 

PANCREAS,  autolysis  of  nucleic  acids 

in,  30 
removal     of,     effect    on    erepsine, 

126 
extract    of,   causing    glycolysis    in 

muscles,  66 
v.  Glycosuria 
Pelargonic  acid,  104 
Pentoses  in  nucleic  acids,  27 

lactic  acid  from,  41 
Pentosuria,  69 
Peptic  digestion,  amido  acids  formed 

in,  125 

action  on  polypeptides,  132 
Pfliiger,  pancreatic  glycosuria,  37 
on   origin   of  sugar    from  fat  and 

proteid,  114  seq. 
criticism  of  Voit  on  fat  formation, 

87 
Phenyl  acetic  acid  series,  fate   of,  in 

body,  105 
Phenyl  alanine,  7 

not    formed    in    tryptic    digestion, 

132 

effect  on  urea  output,  161 
liberated  by  pepsine,  169 
effect  on  alcaptonuria,  195 
Phlorrhizine,  acetonuria  in  dogs,  in 
glycosuria,  fat  in,  113 
indicanuria,  177 
Phosphorus  poisoning  and  fatty  heart, 

89,  118 

and  amido  acids  in  urine,  139 
autolysis  of  liver  in,  146 
Phosphotungstic  acid,  6 
Phylloporphyrine,  170 
Pneumonic  exudation,  absorption  of, 
146 


204 


INDEX 


Polypeptides,  5 

unattacked  by  trypsine,  131,  169 
Proline,  169 
not    formed    in    tryptic    digestion, 

132 

Protamines,  123,  166 
Proteid,  colour  tests,  3 
amount  of,  in  animal  body,  35 
as    source    of   sugar,   37   seq.,    115 

seq. 

fat,  86,  164 
lactic  acid,  60 
acetone  bodies,  106 
synthesis   of,   in   animals,   123,   133 

seq. 

digestion  of,  124  seq. 
specificity  of  proteids  in  blood,  123 
heat  equivalents  of  cleavage  pro- 
ducts, 129 

rate  of  catabolism  in  tissues,  149 
metabolism  on  proteid  diet,  156 
"circulating,"  and  " tissue "  proteid 

controversy,  158 
minimum  intake,  160 
Purine,  178  seq. 

as  source  of  pyrimidine  bases,  181 
synthesis,  182,  187 
Pyridine  derivatives  related  to  trypto- 

phane,  176 
Pyrimidine,  178,  180 
Pyrrhol  and  derivatives,  168 
Pyrrholidine    carboxylic    acid.       See 

Proline 
Pyruvic  aldehyde,  precursor  of  lactic 

acid,  56 
in  formation  of  imidazol,  179 

QUINOLINE  derivatives  from   trypto- 
phane,  176 

RANCIDITY,  106 

Respiratory  quotient  in  hybernation, 

112 
Reversible  zymolysis,  23,  74 


Ricinoleic  acid,  78 

Rigor  mortis,  lactic  acid  in,  59 

SACCHARINIC  ACID  from  glucose,  43 
Salmon,  metabolism  of,  in  fresh  water, 

143 

Salvioli's  perfusion  experiments,  134 
San dmeyer's  operation,  114 
Saponification  value  of  fats,  106 
Scatol,  scatol  carboxylic,    and  scatol 

acetic  acids,  173 
Seeds,  germinating,  oil  in,  106,  112 

proteolysis  in,  142 
Serine,  7 

Soaps,  toxicity  of,  75 
Spleen,  oxidation  of  oxypurine  bases 

in,  1 86 

Starvation,  metabolism  in,  97 
acetonuria  in,  108 
causes  no  acetonuria  in  dogs,  1 1 1 
autolysis  in,  142,  156 
Stomach   synthesis    of   proteid    from 

peptone  in,  135 

Succinic  acid  from  haematine,  172 
Succus  entericus,  erepsine  in,  126 
Sugar  decomposition  in  muscles,  66 
effect  of  alkalies  on,  25,  51 
fermentation  of,  52  seq. 
converted  into  fat,  1 1 2  seq. 
derived  from  fat,  1 1 2 

proteid,  37  seq.,  115  seq. 
amount  of,  in  blood,  36 
output  in  diabetes,  36 
Sulphur,    excretion    of,    on    different 

diets,  162 

Sweetbreads,    effect    on    endogenous 
uric  acid,  189 

TARTRONIC  ACID  in  synthesis  of  uric 

acid,  1 88 
Taurine,  n,  148 

Tetra-oxyamido-caproic  acid,  34,  43 
Thiolactic  acid,  175 
Thymine,  180 


INDEX 


205 


Tissues,  nitrogen  metabolism  of,  166 
Tryptic  digestion,  125 

peptides  resisting,  131  seq. 
Tryptophane,  6,  173,  177,  198 
Tyrosine,  heat  equivalent  of,  128 

in  peptic  digestion,  125 

effect  on  urea  output,  161 

and  homogentisic  acid,  195 

URACIL,  180 
Uranyl  acetate,  191 
Urea  synthesis,  10 

in  acetonuria,  109 

in  blood  during  digestion,  140 

excretion  not  a  measure  of  proteid 

combustion,  155 
after  proteid  meal,  156-7 
on  diet  free  from  proteid,  162 

in  uric  acid  synthesis,  187 
Uric  acid,  182  seq. 

synthesis,  162,  187  seq, 

exogenous,  183 

endogenous,  184 

destruction  in  body,  190 


VALERIANIC  ACID  in  metabolism,  103 
Virchow  on  fatty  degeneration,  87 
Voit,  formation  of  fat  from  proteid,  86 

WORK  and  oxygen  consumption,  100 
See  Muscular  activity. 

XANTHINE,  methylation  of,  in   dogs, 

1 68 
relation   to   methyl    imidazol,    180, 

182 

Xanthine  oxidase,  186 
Xylose  constitution,  29 
from  glycuronic  acid,  29 
in  nucleic  acids,  69 

YEAST,  fermentation  by  enzymes  in, 

12,  52 
produces  formic  acid,  53 

ZYMASE,  12,  52 

Zuntz,  energy  expenditure  on  respira- 
tion and  circulation,  98 
use  of  fat  in  muscles,  100 


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